The present invention relates to C. elegans insulin-like genes and methods for identifying insulin-like genes. The methods provide nucleotide sequences of C. elegans insulin-like genes, amino acid sequences of their encoded proteins, and derivatives (e.g., fragments) and analogs thereof. The invention further relates to fragments (and derivatives and analogs thereof) of insulin-like proteins which comprise one or more domains of an insulin-like protein. Antibodies to an insulin-like protein, and derivatives and analogs thereof, are provided. Methods of production of an insulin-like protein (e.g., by recombinant means), and derivatives and analogs thereof, are provided. Methods to identify the biological function of a C. elegans insulin-like gene are provided, including various methods for the functional modification (e.g., overexpression, underexpression, mutation, knock-out) of one gene, or of two or more genes simultaneously. Methods to identify a C. elegans gene which modifies the function of, and/or functions in a downstream pathway from, an insulin-like gene are provided.
Citation of references herein shall not be construed as an admission that such references are prior art to the present invention.
Insulin-like proteins are a large and widely-distributed group of structurally-related peptide hormones that have pivotal roles in controlling animal growth, development, reproduction, and metabolism (Blundell and Humbel, 1980, xe2x80x9cHormone families: pancreatic hormones and homologous growth factorsxe2x80x9d, Nature 287:781-787). Consequently, the insulin superfamily has become one of the most intensively investigated classes of peptide hormones. Such hormones have a vast array of uses including, for example, clinical applications in human disease, management of fish and livestock, and the control of agriculturally-important animal pests. At least five different subfamilies of insulin-like proteins have been identified in vertebrates, represented by insulin (Steiner et al., 1989, in Endocrinology, DeGroot, ed., Philadelphia, Saunders, pp. 1263-1289), insulin-like growth factor (IGF, previously termed somatomedin) (Humbel, 1990, xe2x80x9cInsulin-like growth factors I and IIxe2x80x9d, Eur. J. Biochem. 190:445-462), relaxin (Schwabe and Bullesback, 1994, xe2x80x9cRelaxin: structures, functions, promises, and nonevolutionxe2x80x9d, FASEB J. 8:1152-1160), relaxin-like factor (RLF, previously called Leydig cell-specific insulin-like peptide) (Adham al., 1993, xe2x80x9cCloning of a cDNA for a novel insulin-like peptide of the testicular Leydig cellsxe2x80x9d, J. Biol. Chem. 268:26668-26672; Ivell, 1997, xe2x80x9cBiology of the relaxin-like factor (RLF)xe2x80x9d, Reviews of Reproduction 2:133-138), and placentin (also known as early placenta insulin-like peptide, or ELIP) (Chassin et al., 1995, xe2x80x9cCloning of a new member of the insulin gene superfamily (INSL4) expressed in human placentaxe2x80x9d, Genomics 29:465-470).
Insulin superfamily members in invertebrates have been less extensively analyzed than in vertebrates, but a number of different subgroups have been defined. Such subgroups include molluscan insulin-related peptides (MIP-I to MIP-VII) (Smit et al., 1988, xe2x80x9cGrowth-controlling molluscan neurons produce the precursor of an insulin-related peptidexe2x80x9d, Nature 331:535-538; Smit et al., 1995, xe2x80x9cExpression and characterization of molluscan insulin-related peptide VII from the mollusc Lymnaea stagnalisxe2x80x9d, Neuroscience 70:589-596), the bombyxins of lepidoptera (originally referred to as prothoracicotropic hormone or PTTH) (Kondo et al., 1996, xe2x80x9cMultiple gene copies for bombyxin, an insulin-related peptide of the silkmoth Bombyx mori: structural signs for gene rearrangement and duplication responsible for generation of multiple molecular forms of bombyxinxe2x80x9d, J Mol. Biol. 259:926-937), and the locust insulin-related peptide (LIRP) (Lagueux et al., 1990, xe2x80x9ccDNAs from neurosecretory cells of brains of Locusta migratoria (Insecta, Orthoptera) encoding a novel member of the superfamily of insulinsxe2x80x9d, Eur. J. Biochem. 187:249-254). Most recently, putative orthologs of both vertebrate insulin and IGF have been identified in a tunicate (McRory and Sherwood, 1997, xe2x80x9cAncient divergence of insulin and insulin-like growth factorxe2x80x9d, DNA and Cell Biology 116:939-949). This is of significance since tunicates are thought to be the closest living invertebrate relative to the progenitor from which vertebrates evolved (Id.).
Comparison of the primary sequence of insulin superfamily peptides, cDNAs, and genes, as well as the overall conservation of functional and structural domains of insulin-like genes and proteins, lead to the conclusion that existing members of the insulin superfamily evolved from a common ancestral gene (Blundell and Humbel, 1980, Id.). From the extensive sequence divergence evident among known subfamilies of insulin-like proteins, it is assumed that this is an ancient family of regulatory hormones that evolved to control growth, reproduction and metabolism in early metazoans. However, the precise evolutionary origins of this important family remain unclear. Indeed, until now, no bona fide insulin-like proteins or genes had been identified in metazoan orders more primitive than insecta.
There are common structural themes that unite the insulin superfamily of proteins. All insulin-like peptide hormones are synthesized in vivo as precursor proteins having structures that are variations of the structure schematically represented in FIG. 1. Most precursor forms of the insulin superfamily can be divided into four domains, termed Pre, B, C, and A domains, extending in order from the N-terminus to the C-terminus of a precursor polypeptide (see FIG. 1). Precursors of the IGF subfamily are distinguished by having two additional domains at the C-terminal end, termed D and E domains. The N-terminal Pre domain typically contains a hydrophobic signal sequence which directs secretion of the hormone from cells and is removed by the enzymatic action of a signal peptidase during transit into the endoplasmic reticulum (see the asterisk in FIG. 1). Upon folding, the prohormone undergoes additional processing which, in most cases, involves proteolytic cleavage at two sites that excise the C peptide from the mature hormone (see the left-hand and middle arrows illustrated in FIG. 1). These processing steps are mediated by prohormone convertases that cleave at specific positions next to basic residues in the C peptide sequence. As a result, most forms of mature insulin superfamily hormones consist of two polypeptide chains, the A and B peptides, which are covalently joined by disulfide linkages (Sxe2x80x94S) between Cys residues (see Sxe2x80x94S linkages illustrated in FIG. 1). The precise arrangement of Cys residues and disulfide linkages, both between and within the A and B peptides, is highly characteristic of the insulin superfamily of hormones. Nearly all known insulin superfamily members contain six precisely-positioned Cys residues, two in the B chain and four in the A chain, which participate in the formation of three disulfide bonds. Two of these disulfide linkages covalently join the B and A chains (i.e., they form inter-chain bonds), whereas the third disulfide linkage occurs within the A peptide (i.e., as an intra-chain bond) and appears to stabilize a bend in the A chain fold.
The IGF subfamily of hormones has a unique processing pathway. In this subfamily, the connecting C peptide is not removed by processing of the prohormone. Instead, a single proteolytic cleavage event removes the C-terminal E domain (see the right-hand arrow illustrated in FIG. 1). Consequently, mature hormones of the IGF subfamily contain a single polypeptide chain with contiguous B, C, A, and D domains. Despite this difference in proteolytic processing, the disulfide bonding pattern between Cys residues in the IGF subfamily is identical to that of other superfamily members.
In summary, FIG. 1 illustrates the structural organization of precursor forms of the insulin superfamily of hormones. The different domains that make up precursor forms of insulin-like hormones are represented as boxes labeled Pre, B, C, A, D, and E, extending from the N-terminus (left) to the C-terminus (right) of the nascent polypeptide chain, respectively. Domains that may remain in a mature hormone are represented as unshaded boxes (the B, A, and D peptide domains) or as lightly hatched (the C or xe2x80x9cconnectingxe2x80x9d peptide domain). By contrast, domains that are removed during proteolytic processing are represented as shaded (the Pre peptide domain) or as hatched (the E peptide domain). IGF hormones are unique in having D and E peptide domains; these domains are represented as smaller boxes in FIG. 1. Cleavage sites utilized by proteases during proteolytic processing (i.e., protein maturation) are indicated below the boxes. The asterisk marks the position of cleavage by signal peptidase. Arrows indicate cleavage sites by prohormone convertases. Disulfide bonds (Sxe2x80x94S) are represented above the boxes with lines indicating connections between covalently-bonded Cys residues.
Since the A and B peptide domains constitute common structural segments among all mature insulin superfamily hormones, it is not surprising that these domains are the most highly conserved at the primary sequence level. Even among closely-related members of this superfamily, the domains removed by proteolytic processing (i.e., Pre, C and E domains) can differ extensively in amino acid sequence composition (McRory and Sherwood, 1997, Id.; Murray-Rust et al., 1992, xe2x80x9cStructure and evolution of insulins: Implications for receptor bindingxe2x80x9d, BioEssays 14:325-331), in marked contrast to the A and B peptides. Much of the amino acid sequence conservation within the A and B peptide domains reflects residues that play key roles in forming the secondary and tertiary structural elements that are characteristic of the insulin superfamily fold. Aligned sequences of A and B peptide domains from diverse insulin superfamily members are depicted in FIG. 2. This alignment serves to highlight the arrangement of conserved amino acid positions and their relationship to the overall folding pattern of the protein. The three dimensional structures of a number of different insulin superfamily proteins have been determined. Such superfamily proteins include insulin (Hua et al., 1991, xe2x80x9cReceptor binding defined by a structural switch in a mutant human insulinxe2x80x9d, Nature 354:238-241), relaxin (Eigenbrot et al., 1991, xe2x80x9cX-ray structure of human relaxin at 1.5 angstromsxe2x80x9d, J. Mol. Biol. 221:15-21), IGF (Cooke et al., 1991, xe2x80x9cSolution structure of human insulin-like growth factor I: a nuclear magnetic resonance and restrained molecular dynamics studyxe2x80x9d, Biochemistry 30:5484-5491), and bombyxin (Nagata et al., 1995, xe2x80x9cThree-dimensional solution structure of bombyxin-II, an insulin-like peptide of the silkmoth Bombyx mori: structural comparison with insulin and relaxinxe2x80x9d, J. Mol. Biol. 253:749-758). The detailed geometry of amino acid side chains in these structures, as well as common secondary and tertiary structural themes, have provided valuable clues about the forces that promote the formation of the characteristic insulin fold. Common features of the main chain fold of insulin-like structures consist of the following: (1) two helices within the A chain joined by a loop; (2) an extended, N-terminal coil within the B chain followed by a tight turn and a central helix; (3) a hydrophobic cluster or xe2x80x9ccorexe2x80x9d that forms an interface between juxtaposed surfaces of the A and B chains; and (4) three disulfide bonds. The common helical regions found in the A and B chains are illustrated in FIG. 2 above the alignment (see xe2x80x9c less than --- greater than xe2x80x9d symbols in FIG. 2).
Beyond the above-described general features of insulin-like structures, there are an number of specific features that are unique to the various subfamilies of insulin-like proteins. Notably, in insulin and IGFs, the main chain following the B peptide central helix forms a tight turn and an extended beta-strand. By contrast, the B chain in both relaxin and bombyxin adopts a fold comprising an extended central helix followed by a coil.
The stereotypical arrangement of Cys residues which participate in disulfide linkages within the A and B chains was noted above. It is striking that the exact number and spacing of Cys residues is nearly invariant among insulin-like proteins (see positions B7, B19, A6, A7, A11 and A20, with respect to the human insulin sequence in FIG. 2). Among over 140 sequenced members of the insulin superfamily, only a few show deviations from the canonical arrangement of Cys residues. Further, when differences in the arrangement do occur, they tend to be relatively minor. For example, in the case of murine relaxin, the last two Cys residues of the A chain are separated by a spacer of 9 amino acids instead of the canonical 8 amino acids (Evans et al., 1993, xe2x80x9cThe mouse relaxin gene: nucleotide sequence and expressionxe2x80x9d, J. Mol. Endocrinol. 10:15-23). Another interesting variation occurs in the molluscan insulin-like protein (MIP-I). MIP-I appears to have two extra Cys residues, one located N-terminal to the conserved Cys residues within the A chain and the other located N-terminal to the conserved Cys residues of the B chain (see FIG. 2) (Smit et al., 1988, xe2x80x9cGrowth-controlling molluscan neurons produce the precursor of an insulin-related peptidexe2x80x9d, Nature 331:535-538). It has been proposed that this extra pair of Cys residues within MIP-I forms an additional disulfide bond between the A and B chains, thus providing further stability to the folded structure of MIP-I (Id.).
The characteristic insulin core that makes up the interface between the A and B chains is composed of a set of side chains whose conserved hydrophobic nature helps stabilize a tight association. The side chains that participate in the core structure correspond to positions A2, A16, A19, B6, B11, B15, and B18 (see FIG. 2). In addition, the A6-A11 and B19-A20 disulfide bonds are enveloped within the core structure. One other highly-conserved residue within the insulin superfamily is that at B8, which is almost always Gly. The unique flexibility of Gly in this position allows the formation of a tight turn between the extended N-terminus of the B chain and the central helix that immediately follows. Gly residues appear to play a similar role in other positions that promote unique structural features of different insulin subfamily folding patterns. For instance, the Gly at position B20 in insulin and IGF appears important in allowing the formation of a tight turn between the central helix and the following beta-strand of the B chain, a hallmark of this subfamily of structures (Blundell et al., 1972, xe2x80x9cInsulin: the structure in the crystal and its reflection in chemistry and biologyxe2x80x9d, Adv. Protein Chem. 26:279-402). Similarly, a Gly at position A10 in relaxins has been shown to be important for the formation of an exceptionally tight turn between the two A chain helices within the folding pattern of this subfamily (Schwabe and Bullesback, 1994, xe2x80x9cRelaxin: structures, functions, promises, and nonevolutionxe2x80x9d, FASEB J. 8:1152-1160).
An intriguing feature of this diverse family of peptide hormones is the nature of receptor-ligand recognition and the structural basis of its specificity. Although no structures have yet been solved for insulin superfamily receptor-ligand complexes, the issue has been explored through mutational analysis and structure-activity studies of a number of insulin superfamily hormones. The collected results of studies of insulin, relaxin and bombyxin have led to the hypothesis that a common surface is employed by these hormones for receptor-ligand interaction, composed of the central portion of the B chain and the A chain N- and C-termini (Hua et al., 1991, Id.; Blundell et al., 1972, Id.; Murray-Rust et al., 1992, Id.; Nagata et al., 1995, Id.; Bullesbach et al., 1996, xe2x80x9cChemical synthesis of a zwitterhormon, insulaxin, and of a relaxin-like bombyxin derivativexe2x80x9d, Biochemistry 35: 9754-9760; Kristensen et al., 1997, xe2x80x9cAlanine scanning mutagenesis of insulinxe2x80x9d, J. Biol. Chem. 272:12978-12983; Schaffer, 1994, xe2x80x9cA model for insulin binding to the receptorxe2x80x9d, Eur. J. Biochem. 221:1127-1132).
It appears that insulin and relaxin utilize other structural features for receptor recognition beyond these common elements, specifically, the C-terminus of the B chain in insulin and IGF, and the extended A chain N-terminal helix in relaxin (Nagata et al., 1995, Id.; Bullesbach et al., 1996, Id.; Kristensen et al., 1997, Id.). Clearly, it is the precise nature of specific amino acid side chains within the receptor recognition surface that contribute to the affinity and specificity of receptor binding. In this regard, a comparison of the residues implicated in receptor recognition for insulin versus relaxin is informative since these two hormones associate with distinct receptor molecules with no evidence for cross-recognition (Rawitch et al., 1980, xe2x80x9cRelaxin-insulin homology: predictions of secondary structure and lack of competitive bindingxe2x80x9d, Int. J. Biochem. 11:357-362).
Residues implicated in insulin receptor recognition include GlyA1, IleA2, ValA3, LeuA13, TyrA19 and AsnA21 on the A chain and ValB12, TyrB16, LeuB17, PheB24, PheB25, and TyrB26 on the B chain (see FIG. 2). A striking feature of this constellation of side chains is that they are largely hydrophobic in character, particularly through the B chain central helix and beta-strand. It is significant that, within the IGF-I sequence, most of the same positions are occupied by either identical or closely-related amino acids to those found in insulin (see FIG. 2). This is consistent with the observation that, although insulin and IGF-I preferentially associate with distinct receptor molecules, there is still measurable cross-recognition by the receptors. Such cross-recognition is believed to be of physiological significance in vivo, perhaps permitting crosstalk between signals controlling growth and metabolism (Humbel, 1990, Id.).
In relaxin, by marked contrast, two hydrophilic basic residues have been shown to be critical for receptor recognition. These relaxin residues, ArgB9 and ArgB13 (see FIG. 2), protrude one turn apart from the central B helix (Eigenbrot et al., 1991. Id.). Not surprisingly, this pair of Arg residues at positions B9 and B13 are rather distinctive for the relaxin subfamily hormones within vertebrates. Other residues implicated in human relaxin II-receptor recognition include TyrA(-1), PheA19, ValB12, GlnB15 and IleB16 (Bullesbach and Schwabe, 1988, xe2x80x9cOn the receptor binding site of relaxinsxe2x80x9d, Int. J. Peptide Protein Res. 32:361-367).
In summary, FIG. 2 illustrates conserved structural features of known insulin superfamily members. The aligned sequences of the B and A chain peptide domains are shown for representative insulin superfamily hormones from the following vertebrates and invertebrates: human insulin (Bell et al., 1979, xe2x80x9cNucleotide sequence of a cDNA clone encoding human preproinsulinxe2x80x9d, Nature 29:525-527), human IGF-I (Jansen et al., 1983, xe2x80x9cSequence of cDNA encoding human insulin-like growth factor I precursorxe2x80x9d, Nature 306:609-611), human relaxin 1 (Hudson et al., 1983, xe2x80x9cStructure of a genomic clone encoding biologically active human relaxinxe2x80x9d, Nature 301:628-631, RLF from human (Adham al., 1993, xe2x80x9cCloning of a cDNA for a novel insulin-like peptide of the testicular Leydig cellsxe2x80x9d, J. Biol. Chem. 268:26668-26672), placentin from human (Chassin et al., 1995, xe2x80x9cCloning of a new member of the insulin gene superfamily (INSL4) expressed in human placentaxe2x80x9d, Genomics 29:465-470), bombyxin II from silkworm (Nagasawa et al., 1986, xe2x80x9cAmino acid sequence of a prothoracicotropic hormone of the silkworm Bombyx morixe2x80x9d, Proc. Natl. Acad. Sci. U.S.A. 83:5840-5843), MIP from freshwater snail (Smit et al., 1988, xe2x80x9cGrowth-controlling molluscan neurons produce the precursor of an insulin-related peptidexe2x80x9d, Nature 331:535-538), and LIRP from locust (Lagueux et al., 1990, xe2x80x9ccDNAs from neurosecretory cells of brains of Locusta migratoria (Insecta, Orthoptera) encoding a novel member of the superfamily of insulinsxe2x80x9d, Eur. J. Biochem. 187:249-254). The numbering scheme shown at the bottom of the figure is for residues of the A and B chains relative to residue numbers for human insulin peptide domains. The nearly invariant positions of the six Cys residues that participate in disulfide bonds are boxed. MIP-I is unique in having two extra Cys residues which are also individually boxed in that sequence. Other conserved amino acid positions that play important roles in promoting the common insulin superfamily fold are highlighted by shading of the following residue positions: B6, B8, B11, B15, B18, A2, A16, and A19. Three helical regions that comprise the common insulin fold are marked above the alignments using a xe2x80x9c less than --- greater than xe2x80x9d symbol.
As noted above, five different subfamilies of insulin-like hormones are now recognized in humans: insulin, IGF, relaxin, RLF, and placentin. Two of these subfamilies (i.e., RLF and placentin) have been discovered relatively recently and their actual biological roles and corresponding clinical applications remain to be determined. The other three subfamilies (i.e., insulin, IGF and relaxin) have been studied much more extensively and their roles in regulating growth, differentiation, and metabolism has yielded clinical applications of profound and well-known importance, as described briefly below.
Insulin is the central hormone governing metabolism in vertebrates (reviewed in Steiner et al., 1989, Id.). In humans, insulin is secreted by the beta cells of the pancreas in response to elevated blood glucose levels which normally occur following a meal. The immediate effect of insulin secretion is to induce the uptake of glucose by muscle, adipose tissue, and the liver. A longer term effect of insulin is to increase the activity of enzymes that synthesize glycogen in the liver and triglycerides in adipose tissue. Insulin can exert other actions beyond these xe2x80x9cclassicxe2x80x9d metabolic activities, including increasing potassium transport in muscle, promoting cellular differentiation of adipocytes, increasing renal-retention of sodium, and promoting production of androgens by the ovary. Defects in the secretion and/or response to insulin are responsible for the disease diabetes mellitus, which is of enormous economic significance. Within the United States, diabetes mellitus is the fourth most common reason for physician visits by patients; it is the leading cause of end-stage renal disease, non-traumatic limb amputations, and blindness in individuals of working age (Warram et al., 1995, xe2x80x9cEpidemiology and genetics of diabetes mellitusxe2x80x9d, In Joslin""s Diabetes Mellitus, Kahn and Weir, eds., Philadelphia, Lea and Febiger, pp. 201-215; Kahn et al., 1996, xe2x80x9cGenetics of non-insulin dependent (type-II) diabetes mellitusxe2x80x9d, Annu. Rev. Med. 47:509-531; Kahn, 1998, xe2x80x9cType 2 diabetes: when insulin secretion fails to compensate for insulin resistancexe2x80x9d, Cell 92:593-596). Two basic forms of diabetes mellitus occur in humans: type I or insulin-dependent diabetes, and type II or non-insulin-dependent diabetes. A critical problem in managing diabetic patients comes from the phenomenon of insulin resistance, as well as the compounding long term effects of abnormal insulin levels in these individuals. Beyond its role in diabetes mellitus, the phenomenon of insulin resistance has been linked to other pathogenic disorders including obesity, ovarian hyperandrogenism, and hypertension.
The physiologic effects of insulin are mediated by specific association of the peptide hormone with a cell surface receptor, the insulin receptor (IR), with concomitant activation of a signal transduction pathway in responding tissues. The IR has been well-characterized at the molecular level; it is a member of a large family of tyrosine kinase receptors (Ullrich et al., 1985, xe2x80x9cHuman insulin receptor and its relationship to the tyrosine kinase family of oncogenesxe2x80x9d, Nature 313:756-761). IR signaling has been shown to involve a number of intracellular participants (White and Kahn, 1994, xe2x80x9cThe insulin signalling systemxe2x80x9d, J. Biol. Chem. 269:1-4; Kahn et al., 1998, Id.). These participants include the so-called insulin receptor substrate, or IRS-1, which is phosphorylated by an activated insulin receptor kinase. IRS-1 in turn associates with phosphatidyl-inositol-3-kinase (PI3K). A number of other protein kinases and signaling proteins have been implicated in this signal transduction mechanism and presumably participate in a xe2x80x9ckinase cascadexe2x80x9d that leads to the modification and regulation of a host of intracellular enzymes, structural proteins, and transcription factors. Nonetheless, the precise choreography of events involved in insulin signaling remains vague, and a deeper understanding of such events is likely to have application in surmounting the major clinical problem of insulin resistance. In summary, while clinical issues associated with abnormal insulin levels have raised interest in factors regulating the synthesis, secretion and turnover of insulin, many of the underlying regulatory mechanisms remain to be clarified.
Humans express two forms of the IGF subfamily of insulin-like hormones, termed IGF-I and IGF-II (Humbel, 1990, Id.). These proteins have been found to exert powerful mitogenic effects on a variety of cells and tissues, reflecting their normal physiologic role of promoting growth in developing animals. IGF-I is apparently the primary mediator of growth hormone signaling and, as such, is a major mediator of growth of the skeletal system following birth. IGF-II may have a significant role in fetal growth. Detailed studies with IGF-I, in particular, have led to a variety of significant clinical applications in humans which relate to its growth-promoting and mitogenic properties, including treatment of injuries to the central nervous system, peripheral neuropathy, disorders of the gut, osteoporosis, and congestive heart failure, as well as the acceleration of wound-healing (Gluckman and Nikolics, 1988, xe2x80x9cIGF-1 to improve neural outcomexe2x80x9d, U.S. Pat. No. 5,714,460; Ballard and Read, 1997, xe2x80x9cMethod for treating intestinal diseasesxe2x80x9d, U.S. Pat. No. 5,679,771; Clark et al., 1997, Treatment of congestive heart failurexe2x80x9d, U.S. Pat. No. 5,661,122; Lewis et al., 1997, xe2x80x9cPrevention and treatment of peripheral neuropathyxe2x80x9d, U.S. Pat. Nos. 5,420,112, 5,633,228 and 5,648,335; Burk, 1997, xe2x80x9cComposition and method for the treatment of osteoporosis in mammalsxe2x80x9d, U.S. Pat. No. 5,646,116; Antoniades and Lynch, 1993, xe2x80x9cWound healing using IGF-II and TGFxe2x80x9d, U.S. Pat. No. 5,256,644). Since administration of IGF-I has been shown to increase the growth and size of animals, there are possible applications of this hormone in animal husbandry (Humbel, 1990, Id.). As mentioned above, IGFs can elicit insulin-like effects in muscle and adipose tissue, and there is evidence that IGF-I administration may be useful when administered together with insulin in the treatment of diabetes (MacCuish, 1997, xe2x80x9cTreatment of insulin-resistant diabetesxe2x80x9d, U.S. Pat. No. 5,674,845).
The peptide hormone relaxin was first identified as an active substance in extracts of corpora lutea that induced the separation and relaxation of the pubic symphysis in guinea pigs (Schwabe and Bullesback, 1994, Id.). Thus, it was originally believed that the primary physiologic role of relaxin was one associated with promoting parturition during pregnancy. Subsequent studies have confirmed this role in pregnancy for rodents and ruminants. However, the importance of relaxin to the physiology of normal pregnancy in humans is still somewhat in question (Bani, 1997, xe2x80x9cRelaxin: a pleiotropic hormonexe2x80x9d, Gen. Pharmacol. 28:13-22). Recent studies of relaxin have revealed a more complicated and interesting picture of the spectrum of activities of this peptide hormone. Specifically, relaxin has been found to control growth and differentiation of breast cancer cells in vitro, promote blood vessel dilation, have a chronotropic action on the heart, inhibit histamine release by mast cells, affect pituitary hormone secretion, and regulate fluid balance. Given this array of physiologic effects, it is not surprising that a number of clinical applications of relaxin have been pursued. These therapeutic applications of relaxin in humans have included the treatment of intractable pain caused by the swelling or dislocation of tissues, as well as the treatment of congestive heart failure, bradycardia, and neurodegenerative diseases (Cronin et al., 1992, xe2x80x9cUse of relaxin in cardiovascular therapyxe2x80x9d, U.S. Pat. No. 5,166,191; Cronin et al., 1995, xe2x80x9cUse of relaxin in the treatment of bradycardiaxe2x80x9d, U.S. Pat. No. 5,478,807; Yue, 1998, xe2x80x9cMethod of treating fibromyalgia with relaxinxe2x80x9d, U.S. Pat. No. 5,707,642). Two forms of relaxin, which are encoded by separate genes, have been identified in humans (Hudson et al., 1983, xe2x80x9cStructure of a genomic clone encoding biologically active human relaxinxe2x80x9d, Nature 301:628-631; Hudson et al., 1984, xe2x80x9cRelaxin gene expression in human ovaries and the predicted structure of a human preprorelaxin by analysis of cDNA clonesxe2x80x9d, EMBO J. 3:2333-2339). In contrast to insulin and the IGFs, the specific receptor protein(s) for the relaxins have yet to be characterized at either the DNA or protein sequence level.
Studies of insulin-like molecules in invertebrates have been motivated by the desire to identify proteins which play analogous roles to the well-characterized activities of insulin and IGF in mammals. If such hormone activity existed in invertebrates, it would provide an avenue to target the growth, feeding and reproduction of agriculturally-important pest species. The first invertebrate insulin-like proteins to be discovered were the bombyxins of lepidoptera, and they remain the best characterized (Nagasawa et al., 1986, xe2x80x9cAmino acid sequence of a prothoracicotropic hormone of the silkworm Bombyx morixe2x80x9d, Proc. Natl. Acad. Sci. U.S.A. 83:5840-5843). Bombyxin, as the name implies, was first identified in extracts of adult heads of the silkworm Bombyx mori. Curiously, it was found that bombyxin stimulated prothoracic glands of the heterologous moth Samia cynthia ricini to synthesize and secrete ecdysteroid hormone. However, no prothoracicotropic activity was observed when bombyxin was injected into Bombyx mori, raising questions about its normal function in this organism (Kiriishi et al., 1992, xe2x80x9cComparison of the in vivo and in vitro effects of bombyxin and prothoracicotropic hormone on prothoracic glands of the silkworm, Bombyx morixe2x80x9d, Zool. Sci. 9:149-155). Bombyxin is produced by neurosecretory cells within the brain of the silkworm and released into the hemolymph. Recent studies with synthetic bombyxin have suggested a role in regulating carbohydrate metabolism with some similarities to the function of insulin in mammals. When injected into neck-ligated larvae, bombyxin reduced the concentration of the major hemolymph sugar, trehalose, and caused elevated activity of trehalase in the midgut and muscle (Satake et al., 1997, xe2x80x9cBombyxin, an insulin-related peptide of insects, reduces the major storage carbohydrates in the silkworm Bombyx morixe2x80x9d, Comp. Biochem. Physiol. 188B:349-357). Additional studies have revealed a remarkable array of bombyxin genesxe2x80x94over 30 separate bombyxin genes have now been identified in the haploid genome of the silkworm (Kondo et al., 1996, Id.). The bombyxin genes are organized in clusters, and sequence comparisons have led to the categorization of six different gene subtypes. Thus far, all of the bombyxin genes appear to be specifically expressed within four pairs of medial neurosecretory cells in the brain of the silkworm.
DNA-based approaches have been used to isolate insulin-like genes from other invertebrate species, including the LIRP gene from the locust and the MIP-I through MIP-VII series of genes from the freshwater snail (Smit et al., 1998, xe2x80x9cTowards understanding the role of insulin in the brain: lessons from the insulin-related signaling systems in the invertebrate brainxe2x80x9d, Prog. Neurobiol. 54:35-54). The biological function of these other invertebrate superfamily members remains largely uncharacterized. One common theme is that the major site of expression of locust and snail invertebrate insulin-like hormones is in the central nervous system, particularly neurosecretory cells, as has also been observed for the bombyxins of lepidoptera. In the freshwater snail, the cerebral light-green cells, which are the main cells that express the MIP proteins, have been associated with endocrine functions that control glycogen metabolism and the regulation of growth of soft body parts and the shell (Smit et al., 1988, Id.).
Important issues raised in the preceding discussion regarding the biological function, regulation, and signaling mechanisms of insulin superfamily hormones could best be addressed if these pathways could be analyzed using model genetic organisms. In particular, the facile genetic tools currently available in two model organisms, the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, have proven to be of enormous utility in defining the biological function of genes through mutational analysis, as well as for identifying the components of biochemical pathways conserved during evolution with large-scale, systematic genetic screens (Scangos, 1997, xe2x80x9cDrug discovery in the postgenomic eraxe2x80x9d, Nature Biotechnol. 15:1220-1221; Miklos and Rubin, 1996, xe2x80x9cThe role of the genome project in determining gene function: insights from model organismsxe2x80x9d, Cell 86:521-529). Key discoveries regarding constituents of a number of important human disease pathways, such as the Ras pathway and the pathway controlling programmed cell death, first came from genetic analysis of pathways known to have an evolutionary relation in Drosophila and C. elegans, and later shown to have direct relevance to human biology (Yuan et al., 1993, xe2x80x9cThe C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzymexe2x80x9d, Cell 75:641-652; Therrien et al., 1995, xe2x80x9cKSR, a novel protein kinase required for RAS signal transductionxe2x80x9d, Cell 83:879-888; Karim et al., 1996, xe2x80x9cA screen for genes that function downstream of Ras1 during Drosophila eye developmentxe2x80x9d, Genetics 143:315-329; Kornfeld et al., 1995, xe2x80x9cThe ksr-1 gene encodes a novel protein kinase involved in Ras-mediated signaling in C. elegansxe2x80x9d, Cell 83:903-913; Rubin et al., 1997, xe2x80x9cProtein kinase required for Ras signal transductionxe2x80x9d, U.S. Pat. No. 5,700,675; Steller et al., 1997, xe2x80x9cCell death genes of Drosophila melanogaster and vertebrate homologsxe2x80x9d, U.S. Pat. No. 5,593,879).
Accordingly, it can be anticipated that genetic analysis of pathways involving insulin superfamily hormones in Drosophila and/or C. elegans may yield results of similar importance to human disease. For example, systematic identification of participants in intracellular signaling by insulin-like hormones, or components regulating secretion and turnover of insulin-like hormones, could lead to the identification of drug targets, therapeutic proteins, diagnostics, or prognostics useful for treatment or management of insulin resistance in diabetics. In the realm of applications for the control of agriculturally-important pests, mutational analysis of genes encoding insulin-like hormones in C. elegans or Drosophila could provide the first clear evidence of the precise biological function of this class of hormones in invertebrates, and provide a means to validate potential pesticide targets that are constituents of these signaling pathways.
Evidence for evolutionary conservation of insulin-like signaling pathways in invertebrates has come from the identification of apparent homologs of the insulin receptor in both the fruit fly and the nematode (Petruzzelli et al., 1986, xe2x80x9cIsolation of a Drosophila genomic sequence homologous to the kinase domain for the human insulin receptor and detection of the phosphorylated Drosophila receptor with an anti-peptide antibodyxe2x80x9d, Proc. Natl. Acad. Sci. U.S.A. 83:4710-4714; Kimura et al., 1997, xe2x80x9cdaf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegansxe2x80x9d, Science 277:942-946). One insulin receptor homolog has been characterized thus far in Drosophila, termed DIR (Drosophila insulin receptor) (Ruan et al., 1995, xe2x80x9cThe Drosophila insulin receptor contains a novel carboxyl-terminal extension likely to play an important role in signal transductionxe2x80x9d, J. Biol. Chem. 270:4236-4243), which exhibits extensive homology with vertebrate insulin and IGF receptors in both the extracellular ligand-binding domain and the intracellular tyrosine kinase domain. DIR is larger than the human insulin receptor protein due to extensions at both the N- and C-termini of the polypeptide chain. It is interesting that the additional C-terminal segment of the DIR shares sequence features with IRS-1, one of the substrates of the insulin receptor tyrosine kinase in mammals. Genetic analysis of DIR function in Drosophila has revealed that it is an essential gene with an apparent role in the development of the epidermis and nervous system (Fernandez et al., 1995, xe2x80x9cThe Drosophila insulin receptor homologue: a gene essential for embryonic development encodes two receptor isoforms with different signaling potentialxe2x80x9d, EMBO J. 14:3373-3384). The role, if any, that DIR may play in metabolic regulation in Drosophila remains unclear.
Recent discoveries from studies of C. elegans have also led to the identification of components involved in a presumptive insulin signaling pathway. Furthermore, in C. elegans, unlike Drosophila, there are clear connections of this pathway to important aspects of metabolic regulation. This realization has emerged from genetic dissection of the process of dauer larvae formation in the-nematode (reviewed in Riddle and Albert, 1997, xe2x80x9cGenetic and environmental regulation of dauer larva developmentxe2x80x9d, In C. elegans II, Riddle et al., eds., Cold Spring Harbor Press, Plainview, N.Y., pp. 739-768), as described further below.
The dauer stage is an alterative developmental stage that is induced when environmental factors are not adequate to promote successful reproduction in C. elegans. There are a number of behavioral, morphologic and metabolic changes that characterize the dauer stage which promote survival of the organism under unfavorable conditions. For example, dauer larvae remain relatively motionless, stop feeding, remain small in size and are reproductively immature. Further, there is increased deposition of fat, a reduction of TCA cycle flux, increased phosphofructokinase activity and increased flux through the glyoxylate cycle in dauer larvae, indicating increased reliance on glycogen and lipid stores as energy reserves in the dauer state (O""Riordan and Burnell, 1989, xe2x80x9cIntermediary metabolism in the dauer larva of the nematode C. elegans I. Glycolysis, gluconeogenesis, oxidative phosphorylation and the tricarboxylic acid cyclexe2x80x9d, Comp. Biochem. Physiol. 92B:233-238; O""Riordan and Burnell, 1990, xe2x80x9cIntermediary metabolism in the dauer larva II. The glyoxylate cycle and fatty acid oxidationxe2x80x9d, Comp. Biochem. Physiol. 95B:125-130; Wadsworth and Riddle, 1989, xe2x80x9cDevelopmental regulation of energy metabolism in Caenorhabditis elegansxe2x80x9d, Devel. Biol. 132:167-173). Dauer larvae are relatively resistant to detergent, high temperature and oxygen deprivation as compared to normal adults. Remarkably, dauer larvae can live more than four times as long as the normal life span of C. elegans. 
The main environmental cues that control entry into the dauer state are pheromone, food, and temperature. High levels of pheromone (indicative of high population density), low levels of food, and high temperature all favor entry into the dauer stage; reversal of these conditions can induce exit from the dauer stage with resumption of normal organismal development. Clearly, the decision to enter either the dauer pathway or pursue normal development is a major milestone in the life cycle of C. elegans. As such, it likely involves a complex and precise integration of many different physiologic signals. Laser microsurgery has been used to investigate. the role of specific cells and tissues in regulating entry into the dauer state (Bargmann and Horvitz, 1991, xe2x80x9cControl of larval development by chemosensory neurons in Caenorhabditis elegansxe2x80x9d, Science 251:1243-1246). These cell-killing experiments point to a prominent role for amphid neurons which comprise a pair of chemosensory organs on either side of the head. Killing of specific neurons in the amphid causes a dauer constitutive phenotype, implying that the amphids are responsible for producing a dauer-inhibiting neuronal signal during normal development.
The connection between dauer formation in the nematode and insulin signaling has come from the molecular characterization of the daf-2 gene of C. elegans. A daf-2 mutant animal exhibits a dauer constitutive phenotype, and molecular cloning of this gene has revealed that it is a nematode homolog of vertebrate insulin receptors. The physiologic analogy with insulin signaling in vertebrates is that activation of the daf-2 receptor in the nematode corresponds to a fed state, with the activated daf-2 receptor generating a dauer-inhibiting signal that promotes normal development. Conversely, lack of daf-2 receptor activity corresponds to a starved state, with the lack of inhibitory signal in this pathway favoring entry into the dauer stage. Indeed, studies of other components in the daf-2 signaling pathway have revealed further similarities with insulin signaling in humans. Two other genes, age-1 and daf-16, have been placed in the same pathway as daf-2 based on analysis of genetic interactions (Morris et al., 1996, xe2x80x9cA phosphatidyl-inositol-3-OH kinase family member regulating longevity and diapause in Caenorhabditis elegansxe2x80x9d, Nature 382:536-539; Ogg et al., 1997, xe2x80x9cThe Forkhead transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegansxe2x80x9d, Nature 389:994-999; Lin et al., 1997, xe2x80x9cdaf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegansxe2x80x9d, Science 278:1319-1322). The age-1 gene encodes a nematode homolog of PI3K, and the action of age-1 is required for the propagation of a daf-2 signal, in keeping with the role of PI3K in insulin signaling. Conversely, genetic analysis has shown that the normal role of daf-16 is one of blocking a signal generated by activated daf-2, and daf-16 has been found to encode a homolog of the HNF-3/forkhead family of transcription factors. In this respect, it is relevant that, in humans, there is the suggestion that insulin mediates some of its effects in target cells by blocking the action of HNF-3 (O""Brien et al., 1995, xe2x80x9cHepatic nuclear factor 3- and hormone-regulated expression of the phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein I genesxe2x80x9d, Mol. Cell. Biol. 15:1747-1758).
There is another intriguing aspect to the phenotype of nematodes defective in components of the daf-2 pathway with respect to effects on the life-span of the organism (normally about 14 days). Mutations in daf-2 and age-1 can more than double the life-span of animals, even under conditions that do not induce the formation of dauer larvae, and the extension of life-span caused by daf-2 or age-1 mutations requires the activity of the daf-16 gene (Lin et al., 1997, Id.; Tissenbaum and Ruvkun, 1998, xe2x80x9cAn insulin-like signaling pathway affects both longevity and reproduction in Caenorhabditis elegansxe2x80x9d, Genetics 148:703-717; Larsen et al., 1995, xe2x80x9cGenes that regulate both development and longevity in Caenorhabditis elegansxe2x80x9d, Genetics 139:1567-1583). These findings raise the interesting possibility that detailed genetic analysis of the daf-2 signaling pathway in nematodes, including identification of daf-2 ligands and their regulation, could reveal new therapeutic approaches with application to aging and longevity in humans.
The structural homologies of components of the daf-2 pathway with those involved in insulin signaling in mammals, as well as the function of the daf-2 pathway in controlling metabolism and aging, raise critical questions with respect to further analysis of this pathway and its potential applications. For example, are there, in fact, insulin superfamily hormones in C. elegans? If so, how diverse is the insulin superfamily in C. elegans in terms of structure and function? Further, what specific C. elegans insulin-like protein(s) interact with the daf-2 receptor, or otherwise affect dauer formation or longevity? Finally, how are the synthesis, activity and turnover of insulin-like proteins regulated in C. elegans?
Accordingly, in an effort to answer such questions, an extensive search for insulin-like genes in the genome of C. elegans has been conducted. Further, certain aspects of C. elegans insulin-like gene function have now been characterized herein. The results of this search have revealed a surprisingly large and diverse family of insulin-like genes. Such a large, diverse family is quite unexpected in such a small, morphologically simple organism. Although all of the nematode insulin-like genes identified herein share structural features that are characteristic of the insulin superfamily, there are novel and significant structural elements of the C. elegans insulin-like proteins that have not been found in any previously-characterized members of the superfamily. These new insulin-like genes in C. elegans constitute very useful tools for probing the function and regulation of their corresponding pathways. Systematic genetic analysis of signaling pathways involving insulin-like proteins in C. elegans can be expected to lead to the discovery of new drug targets, therapeutic proteins, diagnostics and prognostics useful in the treatment of diseases and clinical problems associated with the function of insulin superfamily hormones in humans and other animals, as well as clinical problems associated with aging and senescence. Furthermore, analysis of these same pathways using C. elegans insulin-like proteins as tools will have utility for identification and validation of pesticide targets in invertebrate pests that are components of these signaling pathways.
Use of C. elegans insulin-like genes for such purposes has advantages over manipulation of other known components of the nematode daf-2 pathway, such as daf-2, daf-16, and age-1. Use of ligand-encoding C. elegans insulin-like genes will provide a superior approach for identifying factors that are upstream of the receptor in the signal transduction pathway. Specifically, components involved in the synthesis, activation and turnover of insulin-like proteins may be identified. Furthermore, the large number of different insulin-like hormones could provide a means to separate components involved in response to different, specific environmental signals which may not be technically feasible with manipulation of downstream components of the pathway found in target tissues. Further, the diversity of different insulin-like hormones may provide a means to identify new receptor and/or signal transduction systems for insulin superfamily hormones that are structurally different than those that have been characterized to date in either vertebrates or invertebrates. Finally, use of C. elegans as a system for analyzing the function and regulation of insulin-like genes has great advantages over approaches in other organisms due to the ability to rapidly carry out large-scale, systematic genetic screens as well as the ability to screen small molecule libraries directly on whole organisms for possible therapeutic or pesticide use.
The present invention relates to nucleotide sequences of C. elegans insulin-like genes, amino acid sequences of their encoded proteins, and derivatives (e.g., fragments) and analogs thereof. Nucleic acids capable of hybridizing to or complementary to the foregoing nucleotide sequences are also provided. The invention also relates to a method of identifying genes that are modified by, or that participate in signal transduction with, C. elegans insulin-like genes. The invention also relates to derivatives and analogs of C. elegans insulin-like genes which are functionally active, i.e., which are capable of displaying one or more known functional activities associated with a full-length (wild-type) insulin-like protein. Such functional activities include but are not limited to antigenicity (ability to bind, or to compete for binding, to an anti-insulin antibody), immunogenicity (ability to generate antibody which binds to insulin), and ability to bind (or compete for binding) to a receptor for insulin (e.g., C. elegans insulin receptor-like gene daf-2). The invention further relates to fragments (and derivatives and analogs thereof) of an insulin-like protein which comprise one or more domains of an insulin-like protein. Antibodies to an insulin-like protein, derivatives and analogs of an insulin-like protein, are additionally provided. Methods of production of the insulin-like proteins, derivatives and analogs, e.g., by recombinant means, are also provided.
This invention provides a purified C. elegans insulin-like protein comprising an amino acid sequence of SEQ ID NO:198. In one embodiment, the protein comprises amino acid numbers 19 through 103 of SEQ ID NO:198.
This invention provides a purified C. elegans insulin-like protein comprising an amino acid sequence of SEQ ID NO:199. In another embodiment, the protein comprises amino acid numbers 19 through 72 of SEQ ID NO:199.
This invention provides a purified C. elegans insulin-like protein comprising an amino acid sequence of SEQ ID NO:200. In another embodiment, the protein comprises amino acid numbers 17 through 110 of SEQ ID NO:200.
This invention provides a purified C. elegans insulin-like protein comprising amino acid sequence of SEQ ID NO:201. In another embodiment, the protein comprises amino acid numbers 19 through 67 of SEQ ID NO:201.
This invention provides a purified C. elegans insulin-like protein comprising an amino acid sequence of SEQ ID NO:202. In another embodiment, the protein comprises amino acid numbers 20 through 76 of SEQ ID NO:202.
This invention provides a purified C. elegans insulin-like protein comprising an amino acid sequence of SEQ ID NO:203. In another embodiment, the protein comprises amino acid numbers 21 through 120 of SEQ ID NO:203.
This invention provides a purified C. elegans insulin-like protein comprising an amino acid sequence of SEQ ID NO:204. In another embodiment, the protein comprises amino acid numbers 16 through 218 of SEQ ID NO:204.
This invention provides a purified C. elegans insulin-like protein comprising amino acid sequence of SEQ ID NO:205. In another embodiment, the protein comprises amino acid numbers 29 through 107 of SEQ ID NO:205.
This invention provides a purified C. elegans insulin-like protein comprising amino acid sequence of SEQ ID NO:206. In another embodiment, the protein comprises amino acid numbers 23 through 77 of SEQ ID NO:206.
This invention provides a purified derivative of the protein of the above-listed proteins, which derivative is capable of immunospecific binding to an anti-insulin-like protein antibody.
This invention provides a purified derivative of the protein of the above-listed proteins, which derivative displays one or more functional activities of the C. elegans insulin-like protein.
This invention provides a purified fragment of the protein of the above-listed proteins, which fragment displays one or more functional activities of the C. elegans insulin-like protein.
This invention provides a purified fragment of the protein of the above-listed proteins, comprising a domain of the C. elegans insulin-like protein selected from the group consisting of a B peptide domain and an A peptide domain.
This invention provides a chimeric protein comprising the fragment of the above-listed proteins, consisting of at least 6 amino acids fused by a covalent bond to an amino acid sequence of a second protein, which second protein is not a C. elegans insulin-like protein.
This invention provides a chimeric protein comprising the fragment of the above-listed proteins, consisting of at least 6 amino acids fused by a covalent bond to an amino acid sequence of a second protein, which second protein is not a C. elegans insulin-like protein.
This invention provides a chimeric protein of the above-listed proteins, wherein the fragment of the C. elegans insulin-like protein is a fragment capable of immunospecific binding to an anti-insulin-like protein antibody.
This invention provides a chimeric protein of the above-listed proteins, wherein the fragment of the C. elegans insulin-like protein is a fragment capable of immunospecific binding to an anti-insulin-like protein antibody.
This invention provides a purified antibody or an antigen-binding derivative thereof capable of immunospecific binding to the protein of any one of the above-listed proteins and not to an insulin-like protein of another species.
In one embodiment, the antibody is polyclonal. In another embodiment, the antibody is monoclonal.
This invention provides an isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214 and SEQ ID NO:215.
This invention provides an isolated nucleic acid comprising a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205 and SEQ ID NO:206. In one embodiment, the nucleic acid is cDNA. In another embodiment, the nucleic acid is mRNA.
This invention provides an isolated nucleic acid which hybridizes under conditions of high stringency to a second nucleic acid consisting of nucleotide sequence of the nucleic acid of the above-listed nucleotide sequences.
This invention provides a nucleic acid of the above-listed nucleotide sequences which encodes a C. elegans insulin-like protein or a functional derivative thereof.
This invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a functional derivative of an amino acid sequence selected from the group consisting of SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205 and SEQ ID NO:206.
This invention provides an isolated nucleic acid comprising a nucleotide sequence that is antisense to a nucleotide sequence selected from the group consisting of SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214 and SEQ ID NO:215.
This invention provides an isolated nucleic acid comprising a nucleotide sequence that is antisense to a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205 and SEQ ID NO:206.
This invention provides a method of producing a C. elegans insulin-like protein comprising: (a) growing recombinant cell containing a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214 and SEQ ID NO:215 such that the encoded C. elegans insulin-like protein is expressed by the cell; and (b) recovering the expressed C. elegans insulin-like protein. In one embodiment, invention provides a purified C. elegans insulin-like protein produced by this method.
This invention provides a method of producing a C. elegans insulin-like protein comprising: (a) growing a recombinant cell containing a nucleic acid comprising a nucleotide sequence encoding a C. elegans insulin-like protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205 and SEQ ID NO:206 such that the encoded C. elegans insulin-like protein is expressed by the cell; and (b) recovering the expressed C. elegans insulin-like protein. This invention provides a purified C. elegans insulin-like protein produced by the method.
This invention provides a method of identifying a phenotype associated with mutation or abnormal expression of a C. elegans insulin-like protein comprising identifying the effect of a mutated or abnormally expressed C. elegans insulin-like gene which encodes a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205 and SEQ ID NO:206 in a C. elegans animal. In one embodiment, the effect is determined by an assay selected from the group consisting of a dauer formation assay, a developmental assay, an energy metabolism assay, a growth rate assay and a reproductive capacity assay.
This invention provides a method of identifying a phenotype associated with mutation or abnormal expression of a C. elegans insulin-like protein comprising: (a) mutating or abnormally expressing a C. elegans insulin-like gene which encodes a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205 and SEQ ID NO:206 in a C. elegans (b) identifying an effect of the gene mutated or abnormally expressed. In one embodiment, the effect is identified by an assay selected from the group consisting of a dauer formation assay, a developmental assay, an energy metabolism assay, a growth rate assay and a reproductive capacity assay. In another embodiment, the above phenotype gene is mutated or abnormally expressed using a technique selected from the group consisting of EMS chemical deletion mutagenesis, transposon insertion mutagenesis and double-stranded RNA interference.
This invention provides a recombinant cell containing a recombinant nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214 and SEQ ID NO:215.
This invention provides a vector comprising (a) a nucleotide sequence selected from the group consisting of SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214 and SEQ ID NO:215, and (b) an origin of replication. In one embodiment, the vector in which the nucleotide sequence is operably linked to a heterologous promoter.
This invention provides a purified C. elegans insulin-like protein encoded by a nucleic acid capable of hybridizing under conditions of high stringency to a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214 and SEQ ID NO:215.
This invention provides a purified protein consisting of a B peptide domain defined by amino acid numbers 19-77 of SEQ ID NO:198 linked by one or more disulfide bonds to an A peptide domain defined by amino acid numbers 78-103 of SEQ ID NO:198.
This invention provides a purified protein consisting of a B peptide domain defined by amino acid numbers 19-49 of SEQ ID NO:199 linked by one or more disulfide bonds to an A peptide domain:defined by amino acid numbers 50-72 of SEQ ID NO:199.
This invention provides a purified protein consisting of a B peptide domain defined by amino acid numbers 17-82 of SEQ ID NO:200 linked by one or more disulfide bonds to an A peptide domain defined by amino acid numbers 83-110 of SEQ ID NO:200.
This invention provides a purified protein consisting of a B peptide domain defined by amino acid numbers 19-45 of SEQ ID NO:201 linked by one or more disulfide bonds to an A peptide domain defined by amino acid numbers 46-67 of SEQ ID NO:201.
This invention provides a purified protein consisting of a B peptide domain defined by amino acid numbers 21-51 of SEQ ID NO:202 linked by one or more disulfide bonds to an A peptide domain defined by amino acid numbers 52-76 of SEQ ID NO:202.
This invention provides purified protein consisting of a B peptide domain defined by amino acid numbers 21-90 of SEQ ID NO:203 linked by one or more disulfide bonds to an A peptide domain defined by amino acid numbers 91-120 of SEQ ID NO:203.
This invention provides a purified protein consisting of a first B peptide domain defined by amino acid numbers 16-50 of SEQ ID NO:204 linked by one or more disulfide bonds to a first A peptide domain defined by amino acid numbers 51-89 of SEQ ID NO:204, a second B peptide domain defined by amino acid numbers 90-110 of SEQ ID NO:204 linked by one or more disulfide bonds to a second A peptide domain defined by amino acid numbers 111-153 of SEQ ID NO:204, and a third B peptide domain defined by amino acid numbers 154-174 of SEQ ID NO:204 linked by one or more disulfide bonds to a third A peptide domain defined by amino acid numbers 175-218 of SEQ ID NO:204.
This invention provides a purified protein consisting of a B peptide domain defined by amino acid numbers 29-66 of SEQ ID NO:205 linked by one or more disulfide bonds to an A peptide domain defined by amino acid numbers 67-107 of SEQ ID NO:205.
This invention provides a purified protein consisting of a B peptide domain defined by amino acid numbers 23-47 of SEQ ID NO:206 linked by one or more disulfide bonds to an A peptide domain defined by amino acid numbers 48-77 of SEQ ID NO:206.
This invention provides a method of identifying a gene-of-interest as capable of modifying a function of a C. elegans insulin-like gene comprising: (a) constructing a first mutant nematode having a first mutation in the C. elegans insulin-like gene which encodes a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205 and SEQ ID NO:206 and a second mutation in the gene-of-interest; and (b) determining whether the phenotype displayed by the first mutant nematode is different from the phenotype of a second mutant nematode having said first mutation but not said second mutation, in which the displaying of a phenotype by the first mutant nematode that is different from said second mutant nematode identifies the gene-of-interest as capable of modifying the function of the C. elegans insulin-like gene. In one embodiment, the first mutant nematode is produced using a technique selected from the group consisting of EMS chemical deletion mutagenesis, transposon insertion mutagenesis and double-stranded RNA interference. In another embodiment, the phenotype is selected from the group consisting of an altered body shape phenotype, an altered body size phenotype, an altered chemotaxis phenotype, an altered brood size phenotype, an altered egg-laying phenotype, an altered life span phenotype, an altered lipid accumulation phenotype, an altered locomotion phenotype, an altered organ morphogenesis phenotype, an altered thermotaxis phenotype, a dauer constitutive phenotype, a dauer defective phenotype, a lethal phenotype and a sterile phenotype. In yet another embodiment, the altered organ morphogenesis phenotype involves an organ selected from the group consisting of vulva, nervous system, gut and musculature. In fourth embodiment, a nematode having the altered body size phenotype is assayed for activity of a gene affecting body size selected from the group consisting of daf-4, sma-2 and sma-3. In a fifth embodiment, the gene-of-interest is a homolog of an insulin signaling pathway gene from vertebrates. In a sixth embodiment, the gene-of-interest is selected from the group consisting of daf-2, daf-16 and age-1.
This invention provides a C. elegans animal having a first mutation in a C. elegans insulin-like gene comprising a cDNA selected from the group consisting of SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214 and SEQ ID NO:215, and a second mutation in a different gene that is a homolog of an insulin signaling pathway gene from vertebrates.
This invention provides a method of studying a function of a C. elegans insulin-like gene comprising: (a) mis-expressing a wild-type or mutant C. elegans insulin-like gene which encodes a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205 and SEQ ID NO:206 in a transgenic nematode by driving expression with a homologous or heterologous promoter; and (b) detecting a phenotype in said transgenic nematode, so as to study the function of the C. elegans insulin-like gene. In one embodiment, the heterologous promoter driving mis-expression is selected from the group consisting of an hsp 16-2 promoter, an hsp 16-41 promoter, a myo-2 promoter, an hlh-1 promoter and a mec-3 promoter. In another embodiment, the transgenic nematode mis-expressing the C. elegans insulin-like gene further has a mutation in daf-2. In yet another embodiment, the transgenic nematode mis-expressing the C. elegans insulin-like gene is assayed for a change in a phenotype selected from the group consisting of dauer formation and life span.
This invention provides a method of detecting the effect of expression of a C. elegans insulin-like gene on an insulin signaling pathway which encodes a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205 and SEQ ID NO:206 comprising: (a) mutating or abnormally expressing a wild-type C. elegans insulin-like gene in a nematode already having a mutation in the insulin signaling pathway that displays a phenotype-of-interest; and (b) detecting the effect of step (a) on the phenotype-of-interest, so as to detect the effect of expression of the C. elegans insulin-like gene. In one embodiment, the mutation in the insulin signaling pathway is in a gene selected from the group consisting of daf-2, daf-16 and age-1.
This invention provides a method of identifying a molecule that specifically binds to a ligand selected from the group consisting of (i) a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205 and SEQ ID NO:206, (ii) a fragment of the C. elegans insulin-like protein comprising a domain of the protein, and (iii) a nucleic acid encoding the C. elegans insulin-like protein or fragment, the method comprising: (a) contacting the ligand with a plurality of molecules under conditions conducive to binding between the ligand and the molecules; and (b) identifying a molecule within the plurality specifically that binds to the ligand. In one embodiment, the C. elegans insulin-like protein is selected from the group consisting from a signal peptide domain, a pre peptide domain, a pro peptide domain, a B peptide domain, a C peptide domain and an A peptide domain.
This invention provides a recombinant non-human animal in which a C. elegans insulin-like gene which encodes a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205 and SEQ ID NO:206 has been deleted or inactivated by recombinant methods, or a progeny thereof containing the deleted or inactivated gene. In one embodiment, the recombinant non-human animal C. elegans insulin-like gene has been deleted or inactivated by a method selected from the group consisting of EMS chemical deletion mutagenesis, transposon insertion mutagenesis and double-stranded RNA interference.
This invention provides a recombinant non-human animal containing a C. elegans insulin-like transgene which encodes a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205 and SEQ ID NO:206. In one embodiment, the recombinant non-human animal of C. elegans insulin-like transgene is under the control of a promoter that is not the native promoter of the transgene.
This invention provides a purified protein encoded by a first nucleic acid which hybridizes under conditions of high stringency to a second nucleic acid, which second nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:213, SEQ ID NO:214, and SEQ ID NO:215, wherein the protein is characterized as lacking a cleavable C peptide and as having the same number and relative spacing of Cys residues as found in vertebrate insulin-like proteins.
This invention provides a purified protein encoded by a first nucleic acid which hybridizes under conditions of high stringency to a second nucleic acid, which second nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211 and SEQ ID NO:212 wherein the protein is characterized as (a) lacking a cleavable C peptide separating the B and A chains, (b) lacking an intra-chain disulfide bond in the A domain, and (c) having an extra pair of Cys residues relative to vertebrate insulin-like proteins.
This invention provides a purified C. elegans insulin-like protein which the B and A chain domains of the protein are not proteolytically cleaved into separate chains.
This invention provides a method of identifying a molecule that alters the expression level of a C. elegans insulin-like gene corresponding to a cDNA selected from the group consisting of SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214 and SEQ ID NO:215, which method comprises: (a) contacting a transgenic nematode with one or more molecules, said transgenic nematode having a transgene comprising a promoter or enhancer region of genomic DNA from 1 base to 6 kilobases upstream of the start codon of the cDNA operably linked to a reporter gene; and (b) determining whether the level of expression of the reporter gene is altered relative to the level of expression of the reporter gene in the absence of the one or more molecules.
This invention provides a method of identifying a molecule that binds to a promoter or enhancer of a C. elegans insulin-like gene corresponding to a cDNA selected from the group consisting of SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214 and SEQ ID NO:215, which method comprises: (a) contacting a transgenic nematode with one or more molecules, said transgenic nematode having a transgene comprising a promoter or enhancer region of genomic DNA from 1 base to 6 kilobases upstream of the start codon of the cDNA operably linked to a reporter gene; (b) determining whether the level of expression of the reporter gene is altered relative to the level of expression of the reporter gene in the absence of the one or more molecules; (c) contacting the one or more molecules with the promoter or enhancer region of genomic DNA; and (d) identifying the molecule contacted in step (c) that binds to the promoter or enhancer. In one embodiment, the reporter gene encodes green fluorescent protein.
This invention provides a purified C. elegans insulin-like protein comprising an amino acid sequence of SEQ ID NO:158. In one embodiment, the protein of comprises amino acid numbers 30 through 85 of SEQ ID NO:158. This invention provides a purified C. elegans insulin-like protein comprising an amino acid sequence of SEQ ID NO:159. In another embodiment, the protein comprises amino acid numbers 21 through 81 of SEQ ID NO:159. The invention provides a purified C. elegans insulin-like protein comprising an amino acid sequence of SEQ ID NO:160. In another embodiment, the protein comprises amino acid numbers 22 through 83 of SEQ ID NO:160. The invention provides a purified C. elegans insulin-like protein comprising amino acid sequence of SEQ ID NO:161. In another embodiment, the protein comprises amino acid numbers 18 through 73 of SEQ ID NO:161. In another embodiment, the protein comprises a purified derivative of the protein of the above-listed proteins, which derivative is capable of immunospecific binding to an anti-insulin-like protein antibody. In another embodiment, the protein comprises a purified derivative of the above-listed proteins, which derivative displays one or more functional activities of the C. elegans insulin-like protein.
This invention provides a purified fragment of the above-listed proteins, which fragment displays one or more functional activities of the C. elegans insulin-like protein.
This invention provides a purified fragment of the above-listed proteins, comprising a domain of the C. elegans insulin-like protein selected from the group consisting of a B peptide domain and an A peptide domain.
This invention provides a chimeric protein comprising the fragment of the above-listed protein, consisting of at least 6 amino acids fused by a covalent bond to an amino acid sequence of a second protein, which second protein is not a C. elegans insulin-like protein.
This invention provides a chimeric protein comprising the fragment of the above-listed proteins, consisting of at least 6 amino acids fused by a covalent bond to an amino acid sequence of a second protein, which second protein is not a C. elegans insulin-like protein.
This invention provides a chimeric protein of the above-listed proteins, wherein the fragment of the C. elegans insulin-like protein is a fragment capable of immunospecific binding to an anti-insulin-like protein antibody.
This invention provides a chimeric protein of the above-listed proteins, wherein the fragment capable of immunospecific binding to an anti-insulin-like protein antibody further lacks one or more domains of the insulin-like protein.
This invention provides a purified antibody or an antigen-binding derivative thereof capable of immunospecific binding to the above-listed proteins and not to an insulin-like protein of another species. In one embodiment, the antibody is polyclonal. In another embodiment, the antibody is monoclonal.
This invention provides an isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165.
This invention provides an isolated nucleic acid comprising a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161. In one embodiment, the nucleic acid is cDNA. In another embodiment, the nucleic acid is mRNA. In yet another embodiment, the invention provides an isolated nucleic acid which hybridizes under conditions of high stringency to the above-listed nucleic acids.
This invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a functional derivative of the above-listed nucleic acids.
This invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a functional derivative of an amino acid sequence selected from the group consisting of SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161.
This invention provides an isolated nucleic acid comprising a nucleotide sequence that is antisense to a nucleotide sequence selected from the group consisting of SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165.
This invention provides an isolated nucleic acid comprising a nucleotide sequence that is antisense to a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161.
This invention provides a method of producing a C. elegans insulin-like protein comprising: (a) growing a recombinant cell containing a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165 such that the encoded C. elegans insulin-like protein is expressed by the cell; and (b) recovering the expressed C. elegans insulin-like protein. In another embodiment, this invention provides a C. elegans insulin-like protein produced by this method.
This invention provides a method of producing a C. elegans insulin-like protein comprising: (a) growing a recombinant cell containing a nucleic acid comprising a nucleotide sequence encoding a C. elegans insulin-like protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161 such that the encoded C. elegans insulin-like protein is expressed by the cell; and (b) recovering the expressed C. elegans insulin-like protein. This invention provides a purified C. elegans insulin-like protein produced by this method.
This invention provides a method of identifying a phenotype associated with mutation or abnormal expression of a C. elegans insulin-like protein comprising identifying the effect of a mutated or abnormally expressed C. elegans insulin-like gene which encodes a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161 in a C. elegans animal. In one embodiment, the effect is determined by an assay selected from the group consisting of a dauer formation assay, a developmental assay, an energy metabolism assay, a growth rate assay and a reproductive capacity assay.
This invention provides a method of identifying a phenotype associated with mutation or abnormal expression of a C. elegans insulin-like protein comprising: (a) mutating or abnormally expressing a C. elegans insulin-like gene which encodes a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161 in a C. elegans animal; and (b) identifying an effect of the gene mutated or abnormally expressed. In one embodiment, the effect is identified by an assay selected from the group consisting of a dauer formation assay, a developmental assay, an energy metabolism assay, a growth rate assay and a reproductive capacity assay. In another embodiment of the above phenotype identification methods, the gene is mutated or abnormally expressed using a technique selected from the group consisting of EMS chemical deletion mutagenesis, transposon insertion mutagenesis and double-stranded RNA interference.
This invention provides a recombinant cell containing a recombinant nucleic acid containing a nucleotide sequence selected from the group consisting of SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165.
This invention provides a vector comprising (a) a nucleotide sequence selected from the group consisting of SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165, and (b) an origin of replication. In one embodiment, the nucleotide sequence is operably linked to a heterologous promoter.
This invention provides a purified C. elegans insulin-like protein encoded by a nucleic acid capable of hybridizing under conditions of high stringency to a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165.
This invention provides a purified protein consisting of a B peptide domain defined by amino acid numbers 30-62 of SEQ ID NO:158 linked by one or more disulfide bonds to an A peptide domain defined by amino acid numbers 63-85 of SEQ ID NO:158.
This invention provides a purified protein consisting of a B peptide domain defined by amino acid numbers 21-46 of SEQ ID NO:159 linked by one or more disulfide bonds to an A peptide domain defined by amino acid numbers 47-81 of SEQ ID NO:159.
This invention provides a purified protein consisting of a B peptide domain defined by amino acid numbers 22-57 of SEQ ID NO:160 linked by one or more disulfide bonds to an A peptide domain defined by amino acid numbers 58-83 of SEQ ID NO:160.
This invention provides a purified protein consisting of a B peptide domain defined by amino acid numbers 18-50 of SEQ ID NO:161 linked by one or more disulfide bonds to an A peptide domain defined by amino acid numbers 51-73 of SEQ ID NO:161.
This invention provides a method of identifying a gene-of-interest as capable of modifying a function of a C. elegans insulin-like gene comprising: (a) constructing a first mutant nematode having a first mutation in the C. elegans insulin-like gene which encodes a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161 and a second mutation in the gene-of-interest; and (b) determining whether the phenotype displayed by the first mutant nematode is different from the phenotype of a second mutant nematode having said first mutation but not said second mutation, in which the displaying of a phenotype by the first mutant nematode that is different from said second mutant nematode identifies the gene-of-interest as capable of modifying the function of the C. elegans insulin-like gene. In one embodiment, the first mutant nematode is produced using a technique selected from the group consisting of EMS chemical deletion mutagenesis, transposon insertion mutagenesis and double-stranded RNA interference. In another embodiment, the phenotype is selected from the group consisting of an altered body shape phenotype, an altered body size phenotype, an altered chemotaxis phenotype, an altered brood size phenotype, an altered egg-laying phenotype, an altered life span phenotype, an altered lipid accumulation phenotype, an altered locomotion phenotype, an altered organ morphogenesis phenotype, an altered thermotaxis phenotype, a dauer constitutive phenotype, a dauer defective phenotype, a lethal phenotype and a sterile phenotype. In another embodiment, the altered organ morphogenesis phenotype involves an organ selected from the group consisting of vulva, nervous system, gut and musculature. In yet still another embodiment, a nematode having the altered body size phenotype is assayed for activity of a gene affecting body size selected from the group consisting of daf-4, sma-2 and sma-3. In a fifth embodiment, the gene-of-interest is a homolog of an insulin signaling pathway gene from vertebrates. In a sixth embodiment, the gene-of-interest is selected from the group consisting of daf-2, daf-16 and age-1.
This invention provides a C. elegans animal having a first mutation in a C. elegans insulin-like gene comprising a cDNA selected from the group consisting of SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165, and a second mutation in a different gene that is a homolog of an insulin signaling pathway gene from vertebrates.
This invention provides a method of studying a function of a C. elegans insulin-like gene comprising: (a) mis-expressing a wild-type or mutant C. elegans insulin-like gene which encodes a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161 in a transgenic nematode by driving expression with a homologous or heterologous promoter; and (b) detecting a phenotype in said transgenic nematode, so as to study the function of the C. elegans insulin-like gene. In one embodiment, the heterologous promoter driving mis-expression is selected from the group consisting of an hsp 16-2 promoter, an hsp 16-41 promoter, a myo-2 promoter, an hlh-1 promoter and a mec-3 promoter. In one embodiment, the transgenic nematode mis-expressing the C. elegans insulin-like gene further has a mutation in daf-2. In yet another embodiment, the transgenic nematode mis-expressing the C. elegans insulin-like gene is assayed for a change in a phenotype selected from the group consisting of dauer formation and life span.
This invention provides a method of detecting the effect of expression of a C. elegans insulin-like gene on an insulin signaling pathway comprising: (a) mutating or abnormally expressing a wild-type C. elegans insulin-like gene in a nematode already having a mutation in the insulin signaling pathway that displays a phenotype-of-interest; and (b) detecting the effect of step (a) on the phenotype-of-interest, so as to detect the effect of expression of the C. elegans insulin-like gene. In one embodiment, the mutation in the insulin signaling pathway is in a gene selected from the group consisting of daf-2, daf-16 and age-1.
This invention provides a method of identifying a molecule that specifically binds to a ligand selected from the group consisting of (i) a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161, (ii) a fragment of the C. elegans insulin-like protein comprising a domain of the protein, and (iii) a nucleic acid encoding the C. elegans insulin-like protein or fragment, the method comprising: (a) contacting the ligand with a plurality of molecules under conditions conducive to binding between the ligand and the molecules; and (b) identifying a molecule within the plurality that specifically binds to the ligand. In one embodiment, the domain of the C. elegans insulin-like protein is selected from the group consisting of a signal peptide domain, a pre peptide domain, a pro peptide domain, a B peptide domain, a C peptide domain and an A peptide domain.
This invention provides-a recombinant non-human animal in which a C. elegans insulin-like gene which encodes a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161 has been deleted or inactivated by recombinant methods, or a progeny thereof containing the deleted or inactivated gene. In one embodiment, the recombinant non-human animal encoding the C. elegans insulin-like gene has been deleted or inactivated by a method selected from the group consisting of EMS chemical deletion mutagenesis, transposon insertion mutagenesis and double-stranded RNA interference.
This invention provides a recombinant non-human animal containing a C. elegans insulin-like transgene which encodes a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160 and SEQ ID NO:161. In one embodiment, the recombinant non-human animal encoding the C. elegans insulin-like transgene is under the control of a promoter that is not the native promoter of the transgene.
This invention provides a purified protein encoded by a first nucleic acid which hybridizes under conditions of high stringency to a second nucleic acid, which second nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:162, wherein the protein is characterized as lacking a cleavable C peptide separating the B and A chains and as having an extra pair of Cys residues relative to vertebrate insulin-like proteins.
This invention provides a purified protein-encoded by a first nucleic acid which hybridizes under conditions of high stringency to a second nucleic acid, which second nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165, which the protein is characterized as lacking a cleavable C peptide and as having the same number and relative spacing of Cys residues as found in vertebrate insulin-like proteins.
This invention provides a purified protein encoded by a first nucleic acid which hybridizes under conditions of high stringency to a second nucleic acid, which second nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33 and SEQ ID NO:36, which the protein is characterized as (a) lacking a cleavable C peptide separating the B and A chains, (b) lacking an intra-chain disulfide bond in the A domain, and (c) having an extra pair of Cys residues relative to vertebrate insulin-like proteins.
This invention provides a purified C. elegans insulin-like protein which the B and A chain domains of the protein are not proteolytically cleaved into separate chains.
This invention provides a method of identifying a molecule that alters the expression level of a C. elegans insulin-like gene corresponding to a cDNA selected from the group consisting of SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165, which method comprises: (a) contacting a transgenic nematode with one or more molecules, said transgenic nematode having a transgene comprising a promoter or enhancer region of genomic DNA from 1 base to 6 kilobases upstream of the start codon of the cDNA operably linked to a reporter gene; and (b) determining whether the level of expression of the reporter gene is altered relative to the level of expression of the reporter gene in the absence of the one or more molecules.
This invention provides a method of identifying a molecule that binds to a promoter or enhancer of a C. elegans insulin-like gene corresponding to a cDNA selected from the group consisting of SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165, which method comprises: (a) contacting a transgenic nematode with one or more molecules, said transgenic nematode having a transgene comprising a promoter or enhancer region of genomic DNA from 1 base to 6 kilobases upstream of the start codon of the cDNA operably linked to a reporter gene; (b) determining whether the level of expression of the reporter gene is altered relative to the level of expression of the reporter gene in the absence of the one or more molecules; (c) contacting the one or more molecules with the promoter or enhancer region of genomic DNA; and (d) identifying the molecule contacted in step (c) that binds to the promoter or enhancer. In one embodiment, the reporter gene encodes green fluorescent protein.
This invention provides a purified C. elegans insulin-like protein. In one embodiment, the protein comprises an amino acid sequence of SEQ ID NO:1. In another embodiment, the protein comprises an amino acid sequence comprising amino acid numbers 31 through 109 of SEQ ID NO:1. In another embodiment, the protein comprises an amino acid sequence of SEQ ID NO:6. In another embodiment, the protein comprises an amino acid sequence comprising residue numbers 18 through 100 of SEQ ID NO:6. In another embodiment, the protein comprises an amino acid sequence of SEQ ID NO:8. In another embodiment, the protein comprises an amino acid sequence comprising residue numbers 19 through 104 of SEQ ID NO:8. In another embodiment, the protein comprises an amino acid sequence of SEQ ID NO:9. In another embodiment, the protein comprises an amino acid sequence comprising residue numbers 19 through 118 of SEQ ID NO:9. In another embodiment, the protein comprises an amino acid sequence of SEQ ID NO:11. In another embodiment, the protein comprises an amino acid sequence comprising residue numbers 22 through 86 of SEQ ID NO:11. In another embodiment, the protein comprises an amino acid sequence of SEQ ID NO:12. In another embodiment, the protein comprises an amino acid sequence comprising residue numbers 20 through 76 of SEQ ID NO:12. In another embodiment, the protein comprises an amino acid sequence of SEQ ID NO:15. In another embodiment, the protein comprises an amino acid sequence comprising residue numbers through 80 of SEQ ID NO:15. In another embodiment, the protein comprises an amino acid sequence of SEQ ID NO:17. In another embodiment, the protein comprises an amino acid sequence comprising residue numbers 20 through 95 of SEQ ID NO:17. In another embodiment, the protein comprises an amino acid sequence of SEQ ID NO:18. In another embodiment, the protein comprises an amino acid sequence comprising residue numbers 19 through 83 of SEQ ID NO:18. In another embodiment, the protein comprises a purified derivative of the above-listed proteins, which derivative is capable of immunospecific binding to an anti-insulin-like protein antibody. In another embodiment, the protein comprises a purified derivative of the above-listed proteins, which derivative displays one or more functional activities of a C. elegans insulin-like protein.
This invention provides a purified fragment of a C. elegans insulin-like protein, which fragment displays one or more functional activities of the C. elegans insulin-like protein.
This invention provides a purified fragment of a C. elegans insulin-like protein comprising a domain of the C. elegans insulin-like protein selected from the group consisting of a B peptide domain and an A peptide domain. In one embodiment, a molecule comprising the fragment is provided.
This invention provides a chimeric protein comprising a fragment of a C. elegans insulin-like protein consisting of at least 6 amino acids fused by a covalent bond to an amino acid sequence of a second protein, which second protein is not a C. elegans insulin-like protein. In one embodiment, the fragment of the C. elegans insulin-like protein is a fragment capable of immunospecific binding to an anti-insulin-like protein antibody. In another embodiment, the fragment capable of immunospecific binding to an anti-insulin-like protein antibody further lacks one or more domains of the insulin-like protein.
This invention provides a. purified antibody or an antigen-binding derivative thereof capable of immunospecific binding to a C. elegans insulin-like protein and not to an insulin-like protein of another species. In one embodiment, the antibody is polyclonal. In another embodiment, the antibody is monoclonal.
This invention provides an isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:36.
This invention provides an isolated nucleic acid comprising a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:18. In one embodiment, the nucleic acid is cDNA. In another embodiment, the nucleic acid is mRNA. In yet another embodiment, this invention provides an isolated nucleic acid which hybridizes under conditions of high stringency to the above-listed nucleic acids. In yet still another embodiment, the nucleic acid encodes a C. elegans insulin-like protein or a functional derivative thereof.
This invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a functional derivative of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:18.
This invention provides an isolated nucleic acid comprising a nucleotide sequence that is antisense to a nucleotide sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:36.
This invention provides an isolated nucleic acid comprising a nucleotide sequence that is antisense to a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:18.
This invention provides a method of producing a C. elegans insulin-like protein comprising: (a) growing a recombinant cell containing a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:36 such that the encoded C. elegans insulin-like protein is expressed by the cell; and (b) recovering the expressed C. elegans insulin-like protein. This invention provides a C. elegans insulin-like protein produced by this method.
This invention provides a method of producing a C. elegans insulin-like protein comprising: (a) growing a recombinant cell containing a nucleic acid comprising a nucleotide sequence encoding a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:18 such that the encoded C. elegans insulin-like protein is expressed by the cell; and (b) recovering the expressed C. elegans insulin-like protein. This invention provides a C. elegans insulin-like protein produced by this method.
This invention provides a method of identifying a phenotype associated with mutation or abnormal expression of a C. elegans insulin-like protein comprising identifying the effect of a mutated or abnormally expressed C. elegans insulin-like gene in a C. elegans animal. In one embodiment, the effect is determined by an assay selected from the group consisting of a dauer formation assay, a developmental assay, an energy metabolism assay, a growth rate assay and a reproductive capacity assay.
This invention provides a method of identifying a phenotype associated with mutation or abnormal expression of a C. elegans insulin-like protein comprising: (a) mutating or abnormally expressing a C. elegans insulin-like gene in a C. elegans animal; and (b) identifying an effect of the gene mutated or abnormally expressed. In one embodiment, the effect is identified by an assay selected from the group consisting of a dauer formation assay, a developmental assay, an energy metabolism assay, a growth rate assay and a reproductive capacity assay. In another embodiment of the above phenotype identification methods, the gene is mutated or abnormally expressed using a technique selected from the group consisting of EMS chemical deletion mutagenesis, transposon insertion mutagenesis and double-stranded RNA interference.
This invention provides a recombinant cell containing a recombinant nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:36.
This invention provides a vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:36, and an origin of replication. In one embodiment, the nucleotide sequence is operably linked to a heterologous promoter.
This invention provides a purified C. elegans insulin-like protein encoded by a nucleic acid capable of hybridizing under conditions of high stringency to a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:36.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 31-58 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 83-109 of SEQ ID NO:1.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 52-79 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 80-106 of SEQ ID NO:2.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 53-76 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 77-106 of SEQ ID NO:3.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 56-80 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 81-107 of SEQ ID NO:4.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 59-87 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 88-112 of SEQ ID NO:5.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 45-73 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 74-100 of SEQ ID NO:6.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 52-80 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 81-105 of SEQ ID NO:7.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 52-79 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 80-104 of SEQ ID NO:8.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 62-90 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 91-118 of SEQ ID NO:9.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 27-56 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 57-91 of SEQ ID NO:10.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 22-51 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 52-86 of SEQ ID NO:11.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 20-52 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 53-76 of SEQ ID NO:12.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 20-58 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 59-83 of SEQ ID NO:13.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 14-50 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 51-76 of SEQ ID NO:14.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 20-57 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 58-80 of SEQ ID NO:15.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 42-76 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 77-108 of SEQ ID NO:16.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 20-62 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 63-95 of SEQ ID NO:17.
This invention provides a purified protein consisting of a B peptide domain defined by residue numbers 19-50 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 51-83 of SEQ ID NO:18.
This invention provides a method of identifying a gene-of-interest as capable of modifying a function of a C. elegans insulin-like gene comprising: (a) constructing a double mutant nematode having a first mutation in the C. elegans insulin-like gene which encodes a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:18 and a second mutation in the gene-of-interest; and (b) determining whether the phenotype displayed by the double mutant nematode is different from the phenotype of a single mutant nematode having said first mutation but not said second mutation, in which the displaying of a phenotype by the double mutant nematode that is different from said single mutant nematode identifies the gene-of-interest as capable of modifying the function of the C. elegans insulin-like gene. In one embodiment, the double mutant nematode is produced using a technique selected from the group consisting of EMS chemical deletion mutagenesis, transposon insertion mutagenesis and double-stranded RNA interference. In another embodiment, the phenotype is selected from the group consisting of an altered body shape phenotype, an altered body size phenotype, an altered chemotaxis phenotype, an altered brood size phenotype, an altered egg-laying phenotype, an altered life span phenotype, an altered lipid accumulation phenotype, an altered locomotion phenotype, an altered organ morphogenesis phenotype, an altered thermotaxis phenotype, a dauer constitutive phenotype, a dauer defective phenotype, a lethal phenotype and a sterile phenotype. In yet another embodiment, the altered organ morphogenesis phenotype comprises an organ selected from the group consisting of vulva, nervous system, gut and musculature. In yet still another embodiment, the nematode having the altered body size phenotype is assayed for activity of a gene affecting body size selected from the group consisting of daf-4, sma-2 and sma-3. In a fifth embodiment the gene-of-interest displays nucleotide or amino acid sequence similarity to an insulin signaling pathway gene from vertebrates. In a sixth embodiment, the gene-of-interest is selected from the group consisting of daf-2, daf-16 and age-1.
This invention provides a C. elegans animal having a first mutation in a gene encoding a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:36, and a second mutation in a different gene that is the homolog of an insulin signaling pathway gene from vertebrates.
This invention provides a method of studying a function of a C. elegans insulin-like gene comprising: (a) mis-expressing a wild-type or mutant C. elegans insulin-like gene by driving expression with a homologous or heterologous promoter; and (b) detecting a phenotype in a transgenic nematode having the mis-expressed gene, so as to identify the function of the C. elegans insulin-like gene. In one embodiment, the heterologous promoter driving mis-expression is selected from the group consisting of an hsp 16-2 promoter, an hsp 16-41 promoter, a myo-2 promoter, an hlh-1 promoter and a mec-3 promoter. In another embodiment, transgenic animals mis-expressing C. elegans insulin-like genes further carry mutations in daf-2. In yet another embodiment, transgenic animals mis-expressing C. elegans insulin-like genes are assayed for changes in a phenotype selected from the group consisting of dauer formation and life span.
This invention provides a method of detecting the effect of expression of a C. elegans insulin-like gene on an insulin signaling pathway comprising: (a) mutating or abnormally expressing a wild-type C. elegans insulin-like gene in a nematode already having a mutation in the insulin signaling pathway that displays a phenotype-of-interest; and (b) detecting the effect of step (a) on the phenotype-of-interest, so as to detect the effect of expression of the C. elegans insulin-like gene. In one embodiment, the mutation in the insulin signaling pathway is in a gene selected from the group consisting of daf-2, daf-16 and age-1.
This invention provides a method of identifying a molecule that specifically binds to a ligand selected from the group consisting of a C. elegans insulin-like protein, a fragment of the C. elegans insulin-like protein comprising a domain of the protein, and a nucleic acid encoding the C. elegans insulin-like protein or fragment comprising: (a) contacting the ligand with a plurality of molecules under conditions conducive to binding between the ligand and the molecules; and (b) identifying a molecule within the plurality that specifically binds to the ligand. In one embodiment, the domain of the C. elegans insulin-like protein is selected from the group consisting from a signal peptide domain, a pre peptide domain, a pro peptide domain, a B peptide domain, a C peptide domain and an A peptide domain.
This invention provides a recombinant non-human animal in which a C. elegans insulin-like gene has been deleted or inactivated by recombinant methods or a progeny thereof containing the deleted or inactivated gene.
In one embodiment, the C. elegans insulin-like gene has been deleted or inactivated by a method selected from the group consisting of EMS chemical deletion mutagenesis, transposon insertion mutagenesis and double-stranded RNA interference. This invention provides a recombinant non-human animal containing a C. elegans insulin-like transgene.
In one embodiment, the C. elegans insulin-like transgene is under the control of a promoter that is not the native promoter of the transgene.
This invention provides a purified protein encoded by a first nucleic acid which hybridizes under conditions of high stringency to a second nucleic acid, which second nucleic acid comprises a nucleotide sequence of SEQ ID NO:1, wherein the protein is characterized as having a cleavable C peptide separating the B and A chains and an extra pair of Cys residues relative to vertebrate insulin-like proteins.
This invention provides a purified protein encoded by a first nucleic acid which hybridizes under conditions of high stringency to a second nucleic acid, which second nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, wherein the protein is characterized as lacking a cleavable C peptide separating the B and A chains and as having an extra pair of Cys residues relative to vertebrate insulin-like proteins.
This invention provides a purified protein encoded by a first nucleic acid which hybridizes under conditions of high stringency to a second nucleic acid, which second nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:28 and SEQ ID NO:29, wherein the protein is characterized as lacking a cleavable C peptide and as having the same number and relative spacing of Cys residues as found in vertebrate insulin-like proteins.
This invention provides a purified protein encoded by a first nucleic acid which hybridizes under conditions of high stringency to a second nucleic acid, which second nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, wherein the protein is characterized as (a) lacking a cleavable C peptide separating the B and A chains, (b) lacking an intra-chain disulfide bond in the A domain which is characteristic of vertebrate insulin-like proteins, and (c) having an extra pair of Cys residues relative to vertebrate insulin-like proteins.
This invention provides a purified protein encoded by a first nucleic acid which hybridizes under conditions of high stringency to a second nucleic acid, which second nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, wherein the protein is characterized as lacking a cleavable C peptide separating the B and A chains and as having uncharacteristic spacing between Cys residues as compared to vertebrate insulin-like proteins.
In one embodiment of the above proteins lacking a cleavable C peptide, the B and A chain domains of the protein are not proteolytically cleaved into separate chains.
This invention provides a method of identifying a molecule that alters the expression level of a C. elegans insulin-like gene corresponding to a cDNA selected from the group consisting of SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:36, which method comprises: (a) contacting a transgenic nematode with one or more molecules, said transgenic nematode having a transgene comprising a promoter or enhancer region of genomic DNA from 1 base to 6 kilobases upstream of the start codon of the cDNA operably linked to a reporter gene; and (b) determining whether the level of expression of the reporter gene is altered relative to the level of expression of the reporter gene in the absence of the one or more molecules. Further, this invention provides a method of identifying a molecule that binds to a promoter or enhancer of a C. elegans insulin-like gene corresponding to a cDNA selected from the group consisting of SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:36, which method comprises: (a) contacting a transgenic nematode with one or more molecules, said transgenic nematode having a transgene comprising a promoter or enhancer region of genomic DNA from 1 base to 6 kilobases upstream of the start codon of the cDNA operably linked to a reporter gene; (b) determining whether the level of expression of the reporter gene is altered relative to the level of expression of the reporter gene in the absence of the one or more molecules; (c) contacting the one or more molecules with the promoter or enhancer region of genomic DNA; and (d) identifying the molecule contacted in step (c) that binds to the promoter or enhancer. In one embodiment, of the above methods, the reporter gene encodes green fluorescent protein.
This invention provides a purified C. elegans insulin-like protein. In one embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:1. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:1 beginning with residue number 31 and ending with residue number 109. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:2. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:2 beginning with residue number 20 and ending with residue number 106. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:3. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:3 beginning with residue number 16 and ending with residue number 106. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:4. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:4 beginning with residue number 18 and ending with residue number 107. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:5. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:5 beginning with residue number 20 and ending with residue number 112. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:6. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:6 beginning with residue number 18 and ending with residue number 100. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:7. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:7 beginning with residue number 19 and ending with residue number 105. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:8. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:8 beginning with residue number 19 and ending with residue number 104. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:9. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:9 beginning with residue number 19 and ending with residue number 118. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:10. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:10 beginning with residue number 27 and ending with residue number 91. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:11. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:11 beginning with residue number 22 and ending with residue number 86. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:12. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:12 beginning with residue number 20 and ending with residue number 76. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:13. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:13 beginning with residue number 20 and ending with residue number 83. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:14. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:14 beginning with residue number 14 and ending with residue number 76. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:15. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:15 beginning with residue number 20 and ending with residue number 80. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:16. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:16 beginning with residue number 20 and ending with residue number 108. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:17. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:17 beginning with residue number 20 and ending with residue number 95. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:18. In another embodiment, the protein has an amino acid sequence substantially as set forth in SEQ ID NO:18 beginning with residue number 19 and ending with residue number 83.
This invention provides a purified C. elegans insulin-like protein. This invention provides a purified C. elegans insulin-like protein having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18. This invention provides a purified C. elegans insulin-like protein encoded by a nucleic acid capable of hybridizing under conditions of high stringency to a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.
This invention provides a purified derivative or analog of the above-listed proteins. In one embodiment, the purified derivative or analog displays one or more functional activities of an insulin-like protein. In another embodiment, the purified derivative or analog is capable of immunospecific binding to an anti-insulin-like protein antibody.
This invention provides a purified fragment of a C. elegans insulin-like protein comprising a domain of the C. elegans insulin-like protein selected from the group consisting of a B peptide domain and an A peptide domain. In a preferred embodiment, a molecule comprising the purified fragment is provided.
This invention provides a chimeric protein comprising a fragment of a C. elegans insulin-like protein consisting of at least 6 amino acids fused by covalent bond to an amino acid sequence of a second protein, which second protein is not an insulin-like protein. In one embodiment, the fragment of the C. elegans insulin-like protein is a fragment capable of immunospecific binding to an anti-insulin-like protein antibody. In another embodiment, the fragment capable of immunospecific binding to an anti-insulin-like protein antibody further lacks one or more domains of the insulin-like protein.
This invention provides a purified antibody capable of immunospecific binding to a C. elegans insulin-like protein. In one embodiment, the antibody is polyclonal. In another embodiment, the antibody is monoclonal.
This invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:24 and SEQ ID NO:30 (i.e., ZK84.N2 and M04D8.1, respectively; see Table 1). In one embodiment, the nucleic acid is cDNA. In another embodiment, the nucleic acid is genomic DNA. In still another embodiment, the isolated nucleic acid comprises a nucleotide sequence complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO:24 and SEQ ID NO:30 (i.e., ZK84.N2 and M04D8.1, respectively; see Table 1). In yet still another embodiment, the isolated nucleic acid comprises a nucleotide sequence capable of hybridizing under conditions of high stringency to a nucleotide sequence selected from the group consisting of SEQ ID NO:24 and SEQ ID NO:30 (i.e., ZK84.N2 and M04D8.1, respectively; see Table 1).
This invention provides a method of producing a C. elegans insulin-like protein comprising: (a) growing a recombinant cell containing a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36 such that the encoded C. elegans insulin-like protein is expressed by the cell; and (b) recovering the expressed C. elegans insulin-like protein from the cell.
This invention provides a method of identifying a C. elegans insulin-like protein signaling pathway comprising: (a) disrupting a C. elegans insulin-like gene; and (b) identifying the effect of the gene disrupted in step (a) in an assay selected from the group consisting of a dauer formation assay, a developmental assay, an energy metabolism assay, a growth rate assay and a reproductive capacity assay. In one embodiment, the gene is disrupted using EMS chemical deletion mutagenesis. In another embodiment, the gene is disrupted using transposon insertion mutagenesis.
This invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:36, corresponding to F13B12.N, ZK84.N2, ZK1251.N, C06E2.N, C17C3.N, M04D8.1, ZK84.N, T28B8.N and ZC334.N, respectively. In one embodiment, the nucleic acid is cDNA. In another embodiment, the nucleic acid is mRNA. In yet another embodiment, the nucleic acid comprises a nucleotide sequence complementary to the above nucleotide sequences. In yet still another embodiment, the nucleic acid is capable of hybridizing under conditions of high stringency to the above-listed nucleic acids.
This invention provides a method of producing a C. elegans insulin-like protein comprising: (a) growing a recombinant cell containing a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, :SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:36 such that the encoded C. elegans insulin-like protein is expressed by the cell; and (b) recovering the expressed C. elegans insulin-like protein from the cell. In another embodiment, this invention provides a C. elegans insulin-like protein produced by the above method.
This invention provides a method of identifying a phenotype associated with mutation or inactivation of a C. elegans insulin-like protein comprising identifying the effect of an inactivated or mutated insulin-like gene in a C. elegans animal in an assay selected from the group consisting of a dauer formation assay, a developmental assay, an energy metabolism assay, a growth rate assay and a reproductive capacity assay. Further, this invention provides a method of identifying a phenotype associated with mutation or inactivation of a C. elegans insulin-like protein comprising: (a) inactivating or mutating an insulin-like gene in a C. elegans animal; and (b) identifying the effect of the gene inactivated or mutated in step (a) in an assay selected from the group consisting of a dauer formation assay, a developmental assay, an energy metabolism assay, a growth rate assay and a reproductive capacity assay. In one embodiment of these methods, the gene is inactivated or mutated using EMS chemical deletion mutagenesis. In another embodiment of these methods, the gene is inactivated or mutated using transposon insertion mutagenesis. In yet another embodiment of these methods, the gene is inactivated or mutated using double-stranded RNA interference.
This invention provides a recombinant cell containing a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:36, corresponding to F13B12.N, ZK84.N2, ZK1251.N, C06E2.N, C17C3.N, M04D8.1, ZK84.N, T28B8.N and ZC334.N, respectively.
This invention provides a vector containing a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:36, corresponding to F13B12.N, ZK84.N2, ZK1251.N, C06E2.N, C17C3.N, M04D8.1, ZK84.N, T28B8.N and ZC334.N, respectively.
This invention provides a purified C. elegans insulin-like protein encoded by a nucleic acid capable of hybridizing under conditions of high stringency to a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:1, which protein consists of a B peptide domain defined by residue numbers 31-58 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 83-109.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:2, which protein consists of a B peptide domain defined by residue numbers 52-79 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 80-106.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:3, which protein consists of a B peptide domain defined by residue numbers 53-76 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 77-106.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:4, which protein consists of a B peptide domain defined by residue numbers 56-80 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 81-107.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:5, which protein consists of a B peptide domain defined by residue numbers 59-87 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 88-112.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:6, which protein consists of a B peptide domain defined by residue numbers 45-73 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 74-100.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:7, which protein consists of a B peptide domain defined by residue numbers 52-80 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 81-105.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:8, which protein consists of a B peptide domain defined by residue numbers 52-79 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 80-104.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:9, which protein consists of a B peptide domain defined by residue numbers 62-90 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 91-118.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:10, which protein consists of a B peptide domain defined by residue numbers 27-56 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 57-91.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:11, which protein consists of a B peptide domain defined by residue numbers 22-51 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 52-86.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:12, which protein consists of a B peptide domain defined by residue numbers 20-52 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 53-76.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:13, which protein consists of a B peptide domain defined by residue numbers 20-58 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 59-83.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:14, which protein consists of a B peptide domain defined by residue numbers 14-50 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 51-76.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:15, which protein consists of a B peptide domain defined by residue numbers 20-57 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 58-80.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:16, which protein consists of a B peptide domain defined by residue numbers 42-76 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 77-108.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:17, which protein consists of a B peptide domain defined by residue numbers 20-62 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 63-95.
This invention provides a purified C. elegans insulin-like protein having an amino acid sequence substantially as set forth in SEQ ID NO:18, which protein consists of a B peptide domain defined by residue numbers 19-50 linked by one or more disulfide bonds to an A peptide domain defined by residue numbers 51-83.
This invention provides a method of identifying functional redundancy of a C. elegans insulin-like gene in contributing to a phenotype comprising: (a) knocking-out function of more than one insulin-like gene simultaneously; and (b) detecting no change in the phenotype. In one embodiment, knocking-out function is carried out by simultaneous injection of more than one double-stranded RNA derived from each gene using the method of double-stranded RNA interference.
This invention provides a method identifying a gene-of-interest as capable of modifying a function of an insulin-like gene comprising: (a) constructing a double mutant nematode having a mutation in the insulin-like gene and the gene-of-interest; and (b) detecting a phenotype in the double mutant nematode which is different from the phenotype of a single mutant nematode in the insulin-like gene, so as to identify the gene-of-interest as capable of modifying the function of the C. elegans insulin-like gene. the double mutant nematode is produced using a technique selected from the group consisting of EMS chemical deletion mutagenesis, transposon insertion mutagenesis and double-stranded RNA interference. In one embodiment, the phenotype is selected from the group consisting of an altered body shape phenotype, an altered body size phenotype, an altered chemotaxis phenotype, an altered brood size. phenotype, an altered egg-laying phenotype, an altered life span phenotype, an altered lipid accumulation phenotype, an altered locomotion phenotype, an altered organ morphogenesis phenotype, an altered thermotaxis phenotype, a dauer constitutive phenotype, a dauer defective phenotype, a lethal phenotype and a sterile phenotype. In another embodiment, the altered organ morphogenesis phenotype comprises an organ selected from the group consisting of vulva, nervous system, gut and musculature. In yet another embodiment, a nematode having the altered body size phenotype is assayed for activity of a gene affecting body size selected from the group consisting of daf-4, sma-2 and sma-3. In yet still another embodiment, the gene-of-interest displays nucleotide or amino acid sequence similarity to an insulin signaling pathway gene from vertebrates. In yet still another embodiment, the gene-of-interest is selected from the group consisting of daf-2, daf-16 and age-1.
This invention provides a method of identifying a function of an insulin-like gene comprising: (a)mis-expressing a wild-type or mutant insulin-like gene by driving expression with a promoter; and (b) detecting a phenotype in a transgenic nematode having the mis-expressed gene, so as to identify the function of the insulin-like gene. in one embodiment, the promoter driving mis-expression is selected from the group consisting of an hsp 16-2 promoter, an hsp 16-41 promoter, a myo-2 promoter, an hlh-1 promoter and a mec-3 promoter. In another embodiment, transgenic animals mis-expressing insulin-like genes further carry mutations in daf-2. In yet another embodiment, transgenic animals mis-expressing insulin-like genes are assayed for changes in a phenotype selected from the group consisting of dauer formation and life span.
This invention provides a method of detecting the effect of expression of a C. elegans insulin-like gene on an insulin signaling pathway comprising: (a) over-expressing, inactivating or mutating a wild-type C. elegans insulin-like gene; and (b) detecting the effect of step (a) on a phenotype exhibited by a nematode already having a mutation in the insulin signaling pathway, so as to detect the effect of expression of the C. elegans insulin-like gene. In one embodiment, the mutation in the insulin signaling pathway occurs in a gene selected from the group consisting of daf-2, daf-16 and age-1.
This invention provides a method of identifying a molecule that specifically binds to a ligand selected from the group consisting of a C. elegans insulin-like protein, a fragment of the C. elegans insulin-like protein comprising a domain of the protein, and a nucleic acid encoding the C. elegans insulin-like protein or fragment comprising: (a) contacting the ligand with a plurality of molecules under conditions conducive to binding between the ligand and the molecules; and (b) identifying a molecule within the plurality that specifically binds to the ligand. In one embodiment, the domain of the C. elegans insulin-like protein is selected from the group consisting from a signal peptide, a pro peptide, a B peptide domain, a C peptide domain and an A peptide domain.
This invention provides a recombinant non-human animal or an ancestor thereof in which a C. elegans insulin-like gene has been deleted or inactivated. In one embodiment, the C. elegans insulin-like gene has been deleted or inactivated by a method selected from the group consisting of EMS chemical deletion mutagenesis, transposon insertion mutagenesis and double-stranded RNA interference.
This invention provides a recombinant non-human animal containing a C. elegans insulin-like transgene. In one embodiment, the C. elegans insulin-like transgene is under the control of a promoter that is not the natural promoter of the transgene (i.e., the promoter is a heterologous promoter).
This invention provides a C. elegans insulin-like protein (SEQ ID NO:1), wherein the mature protein is characterized as having a cleavable C peptide separating the B and A chains and an extra pair of Cys residues (e.g., F13B12.N).
This invention provides a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, wherein the mature protein is characterized as lacking a cleavable C peptide separating the B and A chains and as having an extra pair of Cys residues (e.g., ZK75.1, ZK75.2, ZK75.3, ZK84.6, ZK84.N2, ZK1251.2, ZK1251.N and C06E2.N).
This invention provides a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:10 and SEQ ID NO:11, wherein the mature protein is characterized as lacking a cleavable C peptide and as having the same number and relative spacing of Cys residues as found in vertebrate insulin-like proteins (e.g., C17C3.4 and C17C3.N).
This invention provides a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, wherein the mature protein is characterized as lacking a cleavable C peptide separating the B and A chains and as having an extra pair of Cys residues and as lacking the intra-chain disulfide bond in the A domain which is characteristic of vertebrate insulin-like proteins (e.g., M04D8.1, M04D8.2, M04D8.3 and ZK84.N).
This invention provides a C. elegans insulin-like protein selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, wherein the mature protein is characterized as lacking a cleavable C peptide separating the B and A chains and as having uncharacteristic spacing between Cys residues as compared to vertebrate insulin-like proteins (e.g., F56F3.6, T28B8.2 and ZC334.N).
This invention provides a C. elegans insulin-like protein, wherein the B and A chain domains of the protein are not cleaved into separate chains (e.g., ZK75.1, ZK75.2, ZK75.3, ZK84.6, ZK84.N2, ZK1251.2, ZK1251.N, C06E2.N, C17C3.4, C17C3.N, M04D8.1, M04D8.2, M04D8.3, ZK84.N, F56F3.6, T28B8.2 and ZC334.N).
This invention provides a C. elegans insulin-like protein (SEQ ID NO:1), wherein the mature protein is characterized as having an excised C peptide and an interchain disulfide bond between Cys residue 52 in the B chain and Cys residue 104 in the A chain (e.g., F13B12.N).